شناسايي و تعيين مقاومت به آنتي بيوتيك Erwinia amylovora عامل بيماري آتشك گلابي درختان ميوه دانه دار در استان اصفهان

چكيده انگليسي :

Identification and Antibiotic Resistance Status of Erwinia amylovora The Causal Agent of Fire Blight on Pome Fruits in Isfahan Province Zahra Hosseini za darya16@yahoo com September 21 2013 Department of Plant Protection Isfahan University of Technology Isfahan 84156 83111 Iran Degree M Sc Language Farsi Supervisor M Bahar Ph D Prof mbahar@cc iut ac ir Abstract Fire blight caused by Erwinia amylovora Burrill Winslowet al 1920 is an important disease of pome fruit trees which has been reported from different rosaceous plants The first symptoms of the disease usually appear in early spring during warm and humid weather Infected flowers and leaves exhibit water soaking wilting shriveling with turning brown the fruits The infected fruits have greasy appearance with often exuding droplets of bacterial ooze For many years E amylovora has been causing outbreaks of fire blight disease in different regions of Iran however the incidence of the disease on apple pear and quince trees in Shahin Shahr Meimeh and Natanz revealed that Esfahan province is also under threat of fire blight In this study the strains of the pathogen were isolated from leaves branches and flowers of infected apple pear and quince trees collected from Isfahan Karaj Khorasan Lorestan and West Azarbaijan Based on biochemical physiological and nutritional properties all of strains were confirmed as E amylovora with similar characteristics as described previously in scientific publications In this study PCR as a rapid sensitive and highly specific method was carried out for identification of E amylovora Using primer pair Ea71A B a fragment of 187 bp was amplified in all strains of E amylovora Also an expected DNA fragment of about 900 1000 bp was produced from the isolates which were subjected to PCR reaction with specific primer pair pEA29 pEB29 corresponding to plasmid pEA29 Genetic comparison of E amylovora strains from Esfahan and other parts of the country by rep PCR BOX and ERIC primer sets did not show any significant genetic variation among the isolates Fingerprinting analysis by NTSYS pc program revealed that all strains were grouped in to four clusters at with similarity level of 90 To examine the sensitivity of strains to antibiotics and copper compound all the strains were tested against different concentration of streptomycin oxytetracyclin and copper sulfate by disc and well diffusion method on nutrient agar and also growth reduction measurement in Nutrient broth None of the strains were resistant to streptomycin oxytetracyclin and copper To examine the best way to detect E amylovora from epiphytic phase a polyclonal antibody raised against strain A isolated from apple in Shahin Shahr and its IgG was purified by Ammonium sulfate precipitation method The specifity of Antibody and IgG was confirmed by Ouchterlony double diffusion test and indirect enzyme linked immunosorbent assay ELISA Comparing several detection methods including Indirect ELISA Direct PCR Bio PCR and Ic PCR to detect E amylovora from asymptomatic leaves of apple sprayed with strain A showed a significant difference in detection capability of the methods Although indirect ELISA was able to detect 104 cfu ml both Ic PCR and Bio PCR were found more sensitive with 10 1 cfu ml detection threshold Since indirect ELISA PCR and Ic PCR are unable to differentiate viable and dead cells in epiphytic population it seems that Bio PCR could be a sensitive and functional technique for the detection of E amylovora By Using Ic PCR method a minimum population of E amylovora was detected in broth culture mixed with high population of saprophytic bacteria Also the method was efficient to detect E amylovora from the surface of leaves of apple 16 days after spraying with suspension of strain A Therefore Bio PCR is recommended as a simple reliable and sensitive method for use in epidemiological studies of E amylovora Key words Fire blight Erwinia amylovora rep PCR ELISA Direct PCR Bio PCR Ic PCR

حسيني، زهرا

شناسايي و تعيين مقاومت به آنتي بيوتيك Erwinia amylovora عامل بيماري آتشك گلابي درختان ميوه دانه دار در استان اصفهان

استرپتومايسين , روش هاي سرولوژيكي