Risk of introduction of quarantine organisms: case Clavibacter michiganensis subsp. sepedonicus

Abstract

Transmission of plant pathogens and pests across country borders has raised an alert in the EU. Some of them having a severe economic impact, leading for taking precautionary measures such as quarantine of plant materials transported across borders. Clavibacter michiganensis subsp. sepedonicus (Cms) is listed as a quarantine organism in EU. This bacterium causes potato ring rot disease and presents a worldwide threat due to crop losses during vegetation and in storage. Potato tubers from import in Serbia were tested for its presence in accordance with the official EU Council Directive. From the stolon end of 200 tubers (consist one sample), the small core of tissue containing vascular tissue was removed and the heel ends were taken and crashed in sterile phosphate buffer, then centrifuged. Re suspended pellet was used for immunofluorescence (IF, Loewe Biochemica GmbH), isolation of bacteria and DNA extraction for Polymerase Chain Reaction (PCR) performed with a pathogen-specific primer set PSA-1/PSA-R. As a positive control Cms reference strain CFBP 3561 was used. Two samples of ware potato originated from Belarus and Russian Federation in 2019 gave positive results for the presence of Cms. Visible internal symptom was observed on several tubers, in form of the vascular ring of tuber when they cut transversely. Bacterial ooze stream when tubers squeezed. The isolation of bacteria was performed from the ooze on Nutrient dextrose agar. Creamy-white, smooth colonies were formed after 3-5 days of incubation at 22 ºC. Restriction fragment length polymorphism (RFLP) of PCR products performed with PSA-1/PSA-R primer pair (502 bp) with enzyme BglII (fragments 282 and 220 bp in size) confirmed that the isolates belong to Cms. Pathogenicity was confirmed on aubergine seedlings, showing typical wilting obtained within 15-20 days after inoculation. Serbia is still free area from Cms pathogen due to rejection of contaminated potatoes from import.