Clavibacter michiganensis subsp. michiganensis, Bacterial Canker of Tomato: 1. Conventional and Molecular Identification

Abstract

In May 2006, tomato leaves with watersoaked areas between leaf veins were detected in glasshouses in Padinska Skela. The early-stage symptoms were followed by rapid desiccation to white and pale brown necrosis and subsequently by wilting of entire tomato plants. In June 2006, symptoms of bacterial canker and wilt were also recorded in many greenhouses in the Jablanica County in southern Serbia. Samples of diseased tomato plants were collected from several tomato-growing regions in Serbia in order to identify the causal agent using conventional identification methods (isolation, pathogenicity and bacteriological determinative tests). Another objective of this study was to confirm the identity of isolated bacterial strains by polymerase chain reaction (PCR method). According to the morphological characteristics observed on NA and NBY media, biochemical characteristics, hypersensitive response in four-o’clock plant leaves and pathogenicity test on tomato seedlings, the investigated strains were identified as C. michiganensis subsp. michiganensis. To confirm the identity of isolated strains two PCR protocols were used. Amplification of expected length DNA fragments, 614 bp and 270 bp, respectively, confirmed that the investigated strains belonged to C. michiganensis subsp. michiganensis.

Tokom maja 2006. godine zapažene su vlažne pege na lišću paradajza gajenom u staklenicima u Padinskoj Skeli. Ovi početni simptomi praćeni su brzim sušenjem lisnog tkiva, pojavom bele i svetlomrke nekroze i uvenućem celih biljaka. U junu 2006. godine, simptomi bakterioznog raka i uvenuća paradajza takođe su zapaženi i u mnogim plastenicima u Jablaničkom okrugu. Uzorci obolelih biljaka paradajza sakupljeni su sa nekoliko lokaliteta u Srbiji, u cilju identifikacije prouzrokovača bolesti korišćenjem konvencionalnih metoda za identifikaciju (izolacija, patogenost i bakteriološki testovi). Drugi cilj istraživanja bio je da potvrdimo identitet izolovanih sojeva bakterija primenom lančane reakcije polimeraze (PCR metod). Prema morfološkim karakteristikama na mesopeptonskoj (MPA) i NBY podlozi, biohemijsko-fiziološkim odlikama, hipersenzitivnoj reakciji na noćurku (Mirabilis jalapa) i patogenim odlikama na rasadu paradajza, izolovani sojevi su identifikovani kao C. michiganensis subsp. michiganensis. Identifikacija izolovanih sojeva bakterija potvrđena je korišćenjem dva PCR protokola. Amplifikovani su DNA fragmenti očekivane dužine, 614 bp i 270 bp, čime je po tvrđeno da testirani sojevi pripadaju bakteriji C. michiganen sis subsp. michiganensis.