In-Depth Characterization of Crown Gall Disease of Tobacco in Serbia

In August 2020, the unusual appearance of crown gall symptoms was observed on the tobacco plants (hybrid PVH2310) grown in fields in the Golubinci (Srem district, Serbia) locality. The causal agent isolated from galls located on tobacco roots formed circular, convex, and glistening light blue colonies, and then dark to olive-green-colored bacterial colonies on a semi-selective D1 medium. Molecular analysis based on multiplex PCR and multi-locus sequence analysis (MLSA) using concatenated sequences of the atpD, dnaK, glnA, and rpoB genes as well as 16S rRNA identified Serbian tobacco isolates such as Agrobacterium tumefaciens (biovar 1). Two duplex PCR methods confirmed the presence of the virD2 and virC genes in tobacco isolates. Pathogenicity tests performed on carrot discs and squash fruits resulted in tumor/gall formation after 12 to 16 days post inoculation, respectively. Pathogenicity was also confirmed on tobacco plants, where isolates caused tumor development 21-25 days after inoculation. API 50 CH generated results regarding the biochemical features of the Serbian tobacco isolates. As A. tumefaciens (biovar 1) as a cause of tobacco crown gall has previously been documented solely in Japan, there is presently no data on its wider occurrence. Therefore, this first detailed investigation of A. tumefaciens isolated from naturally infected tobacco in Serbia will contribute to a better understanding of it at the global level.