New strain of 'Candidatus Phytoplasma ulmi' infecting Ulmus minor and Ulmus laevis in Serbia

Elm yellows (EY) phytoplasma ('Candidatus Phytoplasma ulmi') is the causal agent of a decline in American elms in North America, and in Eurasian elm species and hybrids in Europe (Lee et al., 2004). EY is known to infect different Ulmus species: U. americana, U. minor, U. rubra, U. alata, U. serotina, U. crassifolia and U. chenmoui, showing different symptoms such as stunting, witches' broom, yellowing and general decline of the plants (Griffiths et al., 1999, Marcone et al., 1997). In September 2007 leaves with petioles from eighteen elm trees showing symptoms of discrete leaf yellowing were collected from three different sites in Northeast Serbia near the villages of Srednjevo, Ljubičevo and Å uvajić. From each site six samples were collected. At two sites (Srednjevo and Ljubičevo) the affected plants were of European field elm (U. minor), and at the third site they were of European white elm (U. laevis). Leaves of six symptomless young elm trees (U. minor) collected near Belgrade served as the controls.

Total nucleic acids were extracted from fresh leaf midribs and petioles using the CTAB method (Angelini et al., 2001). Phytoplasma identification was conducted using a nested PCR assay with P1/P7 and F2n/R2 primers on the 16S rRNA gene, followed by RFLP analysis with MseI restriction enzyme. Positive results were obtained in nine symptomatic U. minor samples and five U. laevis samples, with RFLP profiles indicating the presence of phytoplasmas of the 16SrV group. None of the symptomless plants were positive for the presence of phytoplasma. Further characterization was performed by amplifying the ribosomal protein genes l22 and s3 using primers rp(V)F1/rpR1 followed by rp(V)F1A/rp(V)R1A, finally by digestion with MseI (Fig. 1A) and Tsp509I (Fig. 1B) (Lee et al., 2004). RFLP profiles with MseI enzyme showed the presence of EY phytoplasmas of 16SrV-A group, but profiles obtained with Tsp509I enzyme were different from the EY control sample and were more similar to FD-C (16Sr V-C group). Subsequently two of these products, one from U. minor and one from U. laevis, were sequenced (GenBank Acc. No. EU592500, EU592501) and showed identical nucleotide sequence to each other. BLAST analyses showed 99% similarity of these isolates with reference strain EY1^T (AY197675). Nucleotide changes are located in two out of three unique regions of the rpl22-rps3 genes reported by Lee et al. (2004) as being species specific for 'Candidatus Phytoplasma ulmi'.

This is the first report of Elm yellows phytoplasma belonging to rRNA group 16SrV-A infecting elm species in Serbia and of its association with Ulmus laevis. It is also the first evidence of strain differences in 'Candidatus Phytoplasma ulmi' detectable by RFLP analysis of ribosomal protein gene PCR products.