بررسی ثبات واحدهای SSR پلاسمید pEA29 جدایه‌های عامل بیماری سوختگی آتشی

Introduction
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.
Conclusion
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.