Study the Stability of SSR Repeats of pEA29 Plasmid of the Casual Agent of Fire Blight

Document Type : Research Article


Ferdowsi University of Mashhad


Fire blight disease causes by Erwinia amylovora infects a wide variety of rosaceous plants. It was first recorded from pear trees in Karaj in year 1990. After that it was observed in many pear and apple orchards of the country. E. amylovora isolates differed slightly in virulence, symptoms and host range which ca be related to different plasmid content. The presence of an universal plasmid, pEA29, has been observed in majority of E. amylovora strains. Short-sequence DNA repeat with eight nt were repeated 3 to 15 times in the PstI fragment of the pEA29 plasmid. Here, the stability of SSR units and efficiency of this method to categorize strains was checked. For this reseaon two methods including amplification and cloning of whole and part of PstI fragment was done and the sequences were compared to eachother. In addition stability was evaluated based on three treatments including long time propagation, keeping strains in cold situation and re-isolation of infected tissues.
Materials and Methods
In this study 20 strains were purchased from the Iranian Plant Protection Research Institute. Their typical phenotypic tests were examined for all strains. All of then were checked with lateral flow immune chromatography in different serial dilutions. The pathogenicity were aassayed using complete fruit. Direct PCR with A/B primers and nested PCR with AJ75/AJ76 were applied for studied strains. Ten representative strains were selected snf part of PstI fragment amplified using RS1/RS2 primers. The PCR products were purified by QIAquick PCR purification kit (Qiagen, USA), cloned in pGEM-T and were sequenced (Macrogen Inc., Korea). Five of 10 were chosen for stability tests including long time propagation and sub culturing each two week for three months, keeping strains in refrigerator for three months and re-isolation of infected tissues.

Results and Discussion
In phenotypic tests all studies strains were facultative anaerobic growth, oxidase negative, catalase positive and non fluorescent on King's B. The biochemical tests for reduction of nitrate, tween 20 and growth at 36 °C were recorded negative. All E. amylovora strains induced the hypersensitive reaction (HR) on tobacco and pelargonium leaves. All phenotypic tests were agreed with standard references. There is no variation in lab expermints.
Pathogenicity assay were checked using immature pear fruit in two separate treatments. Inoculation caused water soaking, tissue necrosis and sometimes necrosis in pear samples. No symptoms were observed in the negative controls. There is no variation in this assay. It seems that in most cases, the pEA29 plasmid can modify synthesis of amylovoran, levansucrase and finally affect pathogenicity in the host plant with respect to the environment. All strains were check by agri strips (Bioreba, Switzeland, Reinach) in lateral flow immune chromatography to confirm presence of E. amylovora.
All strains of E. amylovora amplified a fragment of the expected size using primers A and B. The accuracy of the direct PCr was evaluated by nested PCR using primers AJ75/AJ76. The PCR products were visualized after electrophoresis on 1.2% agarose gels. A 3kb DNA ladder (Fermentas, Lithuania) was used as a molecular size marker in all experiments.
In order to chracterize SSR units in thi study, part of PstI fragment was amplified by RS1/Rs2 primers. The relevant variability found in the length of the this fragment was explained by a variation in the number of copies of a SSRs of 8 bp, GAATTACA. It is recorded 4 and 8 times in apple and rose plants respectively. Recording the SSRs of 4, 5 and 7 in other hosts including pear, hawthorn and quince may be indicated that under natural conditions, a mixture of E. amylovora strains with different SSR numbers can be caused fire blight. Frequent transffering of bacterial isolats to new nutrient agar medium did not change the SSR units ater three months. Among three testd tretments, keeping in cold weather for at leat three months caused variation in repetitive units in IrGh59 isolates. According to SSR stability some researchers believed that mutation, antibiotic treatment, maintaing in specific suitations may changed the numbers of this units. But our results showed that SSR numbers of several strains remained constant under laboratory conditions.
Sequencing the whole PstI fragment may provide better information about SSR units and the flanking regions. The numbers of SSR are stable under experimental conditions and evaluation of E. amylovora isolaes with this method can apply for strain grouping.


Afunian M.R., Mohammadi M., and Rahimian H. 2000. Phenotypic characterization of Iranian strains of Erwinia amylovora, the causal agent of fire blight disease of pome trees. Iranian Journal of Agricultural Sciences, 31: 463-476.
2- Aldridge P., Metzger M., and Geider K. 1997. Genetics of sorbitol metabolism by Erwinia amylovora and its influence on bacterial virulence. Molecular and General Genetics, 256: 611-619
3- Atanasova I., Kabadjova-Hristova P., Stefanova K., Bogatzevska N., and Moncheva P. 2009. Differentiation of Erwinia amylovora strains from Bulgaria by PCR-RFLP analysis. European Journal of Plant Pathology, 124: 451-456.
4- Barionovi D., Giorgi S., Stoeger A.R., Ruppitsch W., and Scortichini M. 2006. Characterization of Erwinia amylovora strains from different host plants using repetitivesequences PCR analysis, and restriction fragment length polymorphism and short sequence DNA repeat of plasmid pEA29. Journal of Applied Microbiology, 100: 1084-1094.
5- Bereswill S., and Geider K. 1997. Characterization of the rcsB gene from Erwinia amylovora and its influence on exopolysaccharide synthesis and virulence of the fire blight pathogen. Journal of Bacteriology, 179: 1354-61.
6- 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: 3522–3526.
7- Braun-Kiewnick A., Vogelsanger J., Schoch B., Franck L., Holliger E., Duffy B., Altenbach D., Oberhansli T., and Bitterlin W. 2009. Ea AgriStrip- a new rapid test for detection of fire blight. Obst-und Weinbau. 145: 7-10
8- Geider K., Auling G., Jakovljevic V., and Volksch B. 2009. A polyphasic approach assigns the pathogenic Erwinia Strains from diseased pear trees in Japan to Erwinia pyrifoliae. Letters in Applied Microbiology, 48: 324-330.
9- Geier G., Geider K. 1993. Characterization and influence on virulence of the levansucrase gene from the fireblight pathogen Erwinia amylovora. Physiological and Molecular Plant Pathology, 42: 387-404.
10- Jock S., Jacob T., Kim W.S., Hildebrand M., Vosberg H.P., and Geider K. 2003. Instability of short-sequence DNA repeats of pear pathogenic Erwinia strains from Japan and Erwinia amylovora fruit tree and raspberry strains. Molecular Genetics and Genomics, 268: 739-749.
11- Kim W.S., and Geider K. 1999. Analysis of variable short sequence DNA repeats on the 29 kp plasmid of Erwinia amylovora strains. European Journal Plant Pathology, 105: 703-713.
12- Kim W.S., Hildebrand M., Jock S., and Geider K. 2001. Molecular comparison of pathogenic bacteria from pear trees in Japan and the fire blight pathogen Erwinia amylovora. Microbiology, 147: 2951-2959.
13- Lecomte P., Manceau C., Paulin J.P., and Keck M. 1997. Identification by PCR analysis on plasmid pEa29 of isolates of Erwinia amylovora responsible of an outbreak in Central Europe. European Journal Plant Pathology, 103: 91-98.
14- Llop P., Bonaterra A., Pen alver J., and Lopez M.M. 2000. Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material. Applied and Environmental Microbiology, 66: 2071–2078.
15- Llop, P., Barbe, S., and Lopez, M. M. 2012. Functions and origin of plasmids in Erwinia species that are pathogenic to or epiphytically associated with pome fruit trees.Tress, 26:31–46.
16- McGhee G.C., and Jones A.L. 2000. Complete nucleotide sequence of ubiquitous plasmid pEA29 from Erwinia amylovora strain Ea88: gene organization and intraspecies variation. Appllied and Environmental Microbiology, 66: 4897–4907.
17- McGhee G.C., and Sundin G.W. 2007. Thiamin biosynthesis and its influence on exopolysaccaride production: a new component of virulence identified on Erwinia amylovora plasmid pEA29. Acta Horticulturae, 793: 271–277.
18- McManus P.S. , and Jones A.C. 1995. Detection of Erwinia amylovora by nested PCR and PCR-dot blot and reverse blot hybridization. Phytopathology, 85: 618-623.
19- Moarefzade N., Mohammadi M., Sharifi Tehrani A., and Zakeri Z. 2009. Efficiency of some serological and PCR based methods in detection of casual agent of fire blight in fruit trees. Iranian Plant Protection Scienc, 40: 55-64.
20- 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.
21- Moslemkhani C., and Sadeghi L., 2011. Comparision and development of new method for detection of Erwinia amylovora in latent infection plant material. Iran. Journal Plant Pathology, 47: 155-156.
22- Niknejad Kazempour M., Kamran E., and Ali B. 2006. Erwinia amylovora Causing fire blight of pear in the Guilan province of Iran. Journal of Plant Pathology, 88: 113-116.
23- Oh C.S., and Beer S.V. 2005. Molecular genetics of Erwinia amylovora involved in the development of fire blight. Federation of European Microbiological Societies Microbiology Letters, 253: 185-192.
24- Omidvar R., Shamsbakhsh M., and Rahimian H. 2006. Determination Erwinia amylovora features using biochemocal and RAPD methods. Iran Journal Plant Pathology, 42: 673-686.
25- Ruppitsch W., Stöger A., and Keck M. 2004. Stability of short sequence repeats and their application for the characterization of Erwinia amylovora strains. FEMS Microbiology Letters, 234:1-8.
26- Schaad N.W., Jones J.B., Chun W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. APS Press, St. Paul M.N., USA. 158 pp.
27- Schnabel E.L., Jones A.L. 1998. Instability of a pEA29 marker in Erwinia amylovora previously used for strain classification. Plant Disease, 82: 1334-1336.
28- Taghdareh Gh., Baghaee-Ravari S., Moslemkhani C., and Mahdikhani, E. 2014. Evaluation of repeat sequences on plasmid pEA29 of Erwinia amylovora from Iran. European Journal of Plant Pathology, 140:735–744.
29- 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.
30- Van Belkum A., Scherer S., Van Alphen L, and Verbrugh H. 1998. Short-sequence DNA repeats in prokaryotic genomes. Microbiol. Molecular Biology Reviews, 62: 275-293.
31- Van der Zwet T., Bonn W.G. 1999. Recent spread and current worldwide distribution of fire blight. Acta Horticulturae, 489: 167–168.
32- Vanneste J.L. 1995. Erwinia amylovora. PP. 21-41. In: Singh. U.S., R.P. Singh and k. Kohmato (Eds.) Pathogenesis and host specificity in Plant Disease: Histopathological, Biotechnical, Genetic and Molecular Bases.Vol. l. Prokayotes, Progamon Press., Oxford. London.
33- Zhao Y.F., Blumer S. E., and Sundin G.W. 2005. Identification of Erwinia amylovora genes induced during infection of immature pear tissue. Journal of Bacteriology, 187: 8088-8103.