بررسي كارآيي برخي روش‌هاي سرولوژيكي و مبتني بر PCR در تشخيص و رديابي باكتري عامل بيماري آتشك درختان ميوه دانه‌دار

A Study of the Efficiency of some Serological and PCR -Based Methods for the Identification and Detection of Erwinia amylovora, the Causal Agent of Pome Trees Fire Blight

Fire blight, a bacterial disease caused by Erwinia amylovora (Burr.) Winslow et al. 1920 is considered as a serious disease on pome fruits. During recent years, this disease has caused serious damages to the fruit growing industry in Iran. Precise and reliable methods for identification and detection of the pathogen can help in its early control, prevent the disease spread and help eliminate the infected trees in orchards and particularly young trees in the nurseries. In this study, two specific polyclonal antisera were developed and used in double diffusion in agar test and Dot Immunobinding Assay (DIBA) to detect E. amylovora (Ea) cells. As many as 1 x 107 cells per ml from pure culture and as well from infected tissues were detected through DIBA. The sensitivity level was increased to as low as 10 cells using the bacterial enrichment step. The efficiency of serological methods was compared with some published PCR based identification methods, too, in which, a single lkb DNA fragment (pstI) of pEA29 plasmid was amplified in Polymerase Chain Reaction (PCR) by using A and B primer pair. The sensitivity levels of this method in water and tissue extract were 2X10 vs 2xl06 CFU/reaction mixture, respectively. The three mentioned methods were able to identify Ea strains representing various hosts and regions of the country and were specific for Ea, in contrast with other bacterial genera. In Nested-PCR, AJ75 and AJ76 specific primers were used to amplify a single 800- bp fragment. In each reaction mixture, this highly sensitive method could detect as few as one and as many as 100 Ea cells in water and in tissue extract, respectively.