First Report of Crown Gall Caused by Agrobacterium tumefaciens on Euphorbia esula/virgata in Europe.

Hypertrophy and hyperplasia resembling crown galls were found on roots of Euphorbia esula/virgata at a single site in east-central Hungary in 2005. E. esula/virgata, known as leafy spurge in North America, is an invasive species causing substantial economic losses to the value of grazing lands in the Northern Great Plains of the United States and is the target of biological control. E. esula/virgata is widely distributed throughout Eurasia and is found on ditch banks, along roadsides, and in other noncultivated areas in its native range. Large galls on roots resembling crown gall were first noted in 1992 on plants collected for phylogenetic studies from three locations in east-central Hungary. One of these sites was relocated during a 2005 survey and galls were collected from infected plants. Galls were diced and incubated overnight in tubes containing 3 ml of sterile water at room temperature (20 to 25°C). The supernatant was streaked onto plates of potato dextrose agar (PDA), medium 1A, medium 2E, and Roy/Sasser medium. After 7 days, colonies were picked and streaked and subsequently purified on PDA. Of 104 isolates used to inoculate three sunflower plants each (by puncturing roots just below the soil line with a sterile dissecting needle holding a drop of fluid matrix containing bacterial cells), 35 caused galls. Thirty-three isolates were randomly selected from the 104 and used to inoculate three tomato plants each at the soil line. Seventeen caused galls, including two isolates that did not cause galls on sunflower. Finally, none of 20 randomly selected isolates caused galls on kalanchoe plants (Kalanchoe blossfeldiana). Three isolates, which formed the largest galls on sunflower, were used to inoculate five plants of E. esula/virgata growing in a 1:1:1 (peat/sand/Bozeman silt loam) potting mix. The tests were repeated. Galls were visible on inoculated plants within 6 weeks. Diagnostic biochemical tests done prior to and after reisolation indicated that the causal agent was Agrobacterium tumefaciens, which differed from A. rhizogenes in the production of alkali from litmus milk, a positive reaction for the ferric ammonium citrate and 3-ketolactose tests, and negative reactions for tests to detect the production of acid from erythritol and alkali from malonic, l-tartaric, and mucic acid. The three isolates of A. tumefaciens from E. esula/virgata had identical sequences and clustered most closely (99.8 to 99.9% similarity) with five isolates of A. tumefaciens from Tibet and Japan on the basis of cluster analysis using 16S rRNA sequences. Crown gall of E. esula/virgata has also been found in Montana and western North Dakota, and isolates were identified as A. tumefaciens biovars 1 and 2 (1) (the latter is now known as A. rhizogenes). To our knowledge, this the first report of crown gall on E. esula/virgata in Europe. Reference: (1) A. J. Caesar. Plant Dis. 78:796, 1994.