'Candidatus Phytoplasma ulmi' causing yellows in Zelkova serrata newly reported in Italy

The Japanese elms are temperate broad-leaved tree species in the genus Zelkova. This genus includes species that are nowadays distributed in East Asia (three species), Western Asia (one species) and the Mediterranean (two species), while it is absent from North America (Denk and Grimm, 2005). Even though a member of the elm family, the Japanese Elm had no disease or pest problems of significance, including Dutch elm disease. Since June 2006, however, several plants of Zelkova serrata grown in Ancona, Marche region (central eastern Italy), have shown symptoms of chlorosis which involve the whole plant or some of the branches, foliar reddening on one or more branches, attenuation of apical dominance and proliferation of lateral shoots, witches' broom, reduced growth and stunting of the plant.

Leaf samples from symptomatic and symptomless plants were collected and the DNA was extracted using the Plant DNeasy mini kit (Qiagen, Hilden, Germany). A molecular diagnosis was carried out to detect phytoplasma by PCR with universal primers P1/P7 (Seemüller et al., 1998) followed by a nested PCR with specific group primers 16Sr(V)F1/R1 (Lee et al., 1994). All of the five symptomatic samples yielded a PCR product of the expected size (1100 bp), both during 2006 and 2007. This PCR product was purified from the 1% agarose gels using Spin Column Wizard SV Gel and the PCR Clean-Up System (Promega), and eluted with sterile ultrapure water, according to the manufacturer's instructions. Five-µl aliquots of the PCR product were digested with 3 U of restriction endonuclease BfaI (New England BioLabs, Beverly, MA, USA), over night at 37°C. The restriction pattern was characterized by two bands of 650 and 450 bp, identical to those in the reference strain of 'Candidatus Phytoplasma ulmi' (EY, kindly provided by C. Marzachì, IVV-CNR, Torino, Italy) (Lee et al., 2004). The pathogen was not found in samples from symptomless plants. Those plants were inoculated by grafting with samples from infected symptomatic plants, and disease symptoms appeared in the entire plant. Leaf samples from artificially inoculated plants tested positive for the presence of the phytoplasma. The sequenced isolate from Z. serrata showed more than 99% identity with 'Ca. Phytoplasma ulmi' strain EY1^T (GenBank Acc. No. AY197655) (Lee et al., 2004).

Several natural infections of 'Ca. Phytoplasma ulmi' have been described on different species of the genus Ulmus (Lee et al., 2004), although it has not been reported on Zelkova spp. This thus appears to be the first report of 'Ca. Phytoplasma ulmi' infection in symptomatic Z. serrata, in Italy and worldwide.