First Report of Xanthomonas hortorum pv. hederae Causing Bacterial Leaf Spot of Hedera hibernica in Slovenia.

In October 2008, water-soaked spots, each 5 to 10 mm in diameter and surrounded by a pale green halo, were observed on leaves of a single Atlantic ivy plant (Hedera hibernica Kirchn.) out of 89 plants imported from the European Union into a commercial greenhouse in Slovenia. Leaves were surface disinfected, and spots were cut and suspended in 10 mM phosphate buffered saline. From this extract, yellow-pigmented, Xanthomonas-like bacterial colonies were isolated onto nutrient agar, and two colonies (NIB Z 1310 and NIB Z 1312) were each identified as a Xanthomonas sp. based on biochemical tests (oxidase negative; positive for hydrolysis of H₂S, starch, and tributyrin; and positive for acid production from sucrose). Both isolates caused a hypersensitive reaction (1) on leaves of tomato cv. Moneymaker. A repetitive extragenic palindromic sequence (REP)-PCR assay using the BOXA1R primer (3) resulted in highly similar DNA fragment banding patterns (Pearson's correlation: 95% identity) between the two isolates (NIB Z 1310 and NIB Z 1312) and the type strain of Xanthomonas hortorum pv. hederae CFBP 4925 (ICMP 453). Partial sequences of the gyrB gene (DNA gyrase, subunit B) (2) from isolates NIB Z 1310 (Accession No. JF794785; 599 bp) and NIB Z 1312 (Accession No. JF794784; 544 bp), showed identical sequences (100% identity with 100% coverage) to type strain ICMP 453 (Accession No. EU498975.1). The pathogenicity of the two isolates from H. hibernica was confirmed on three plants of H. helix 'Evita' (each 6 months old) for each isolate. Plants were sprayed on the abaxial and adaxial side of leaves with 10 ml of a 48-h suspension of the appropriate isolate with approximately 10⁶ CFU/ml (1), covered individually with plastic bags for 24 h, and incubated under high relative humidity (>80%) with 16 h of daylight at 25°C by day and 20°C by night. Three positive and three negative control plants were inoculated with the type strain of X. hortorum pv. hederae CFBP 4925 and 0.01 M magnesium sulfate buffer, respectively. After 21 days, water-soaked spots with a pale green halo were observed on all plants inoculated with the bacterial strains, including the positive control plants. Colonies isolated from these lesions were identical in morphology and BOX-PCR DNA fragment banding patterns to the original isolates. Negative control plants did not develop symptoms, and colonies similar to X. hortorum pv. hederae were not isolated from these plants. To our knowledge, this is the first report of X. hortorum pv. hederae on H. hibernica in greenhouse production in Slovenia. If the disease spreads, it could reduce quality and marketability of this popular ground cover plant. References: (1) Z. Klement et al. Inoculation of Plant Tissues. In: Methods in Phytobacteriology. Akadémiai Kiadó, Budapest, 1990. (2) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009. (3) J. Versalovic et al. Methods Mol. Cell Biol. 5:25, 1994.