Genetic diagnosis of phytopathogenic Agrobacterium tumefaciens isolates from Cydonia oblonga trees by PCR using 16SrRNA primers

Abstract

Agrobacterium is a plant pathogen causing crown disease and plant tumors in 140 or more dicotyledonous plant species. T u mor-causing agrobacteri a are members of the Rhizobiaceae family of nitrogen-fixing legume symbionts, but are harmful to plants. This study's initial goal was to isolate 25 bacterial isolates from crown tumors that developed on the stems of quince trees in various areas of Mosul. The colonies were selected for tests on the basis of the ir size, shape and color . From these , Gram-negative bacterial isolates were chosen and subjected to the following tests ; catalase, L-tyrosine utilization, citrate utilization, utilization of carboh y drates, lactose, mannitol, H 2 S production, pH range, g rowth at: 28, 35, 40 ºC, g rowth absence, ability to grow without biotine and thiamine, growth and pigmenentation in ferric ammonium citrate . Fi ve isolates (AtCo1, AtCo2, AtCo3, AtCo4, AtCo5) demo n strated their ability to form tumors on carrot discs for periods ranging from 22 to 26 days . Growth on the following selective media for their diagnosis produced positive results ; Mac C onkey agar, D1 medium, Luria- B ertani medium, glucose peptone agar, potato dextrose agar, AMM, chromogenic agar medium. These f ive isolates provided positive diagnostic signs and were designated with the symbols AtCo1, AtCo2, AtCo3 , AtCo4 and AtCo5 , in relation to the quince plant, which is the host plant from which the bact e ria were isolated. All exposures to A. tumefaciens formed tumors on carrot discs, according to the results of an artificial infection test used to determine the pathogenicity of the bacterial isolates. The outcomes of molecular diagnosis using agarose gel electroph o resis for the DNA of A. tumefaciens isolates corroborated these findings. The molecular diagnosis was achieved by amplifying the DNA using the 16S rRNA gene primer and the DNA p olymerase chain reaction (sPCR) technique. This allowed the clear separ a tion of single, shiny bands at the molecular size (1150 bp) in the five isolates, confirming their membership of the genus Agrobact e rium . Both of the Biovar I isolates (AtCo5 and AtCo2) from quince tumors were listed in the gene bank unde r the accession nu m ber PQ106659.

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