First identification of a phytoplasma infecting Cornus sanguinea and Sambucus nigra

L. Filippin*, E. Angelini and M. Borgo

CRA - Centro di ricerca per la viticoltura, 26 viale XVIII Aprile 31015, Conegliano (TV), Italy

*isvbd@ispervit.it

Accepted for publication 17/03/08

A study was performed between 2005 and 2007 to identify the presence of any of the phytoplasmas that normally infect grapevines in Italy (16SrV-C, 16SrV-D and 16SrXII-A) in wild plants in the vicinity of vineyards. This survey included sampling from Cornus sanguinea (36 plants) and Sambucus nigra (32 plants) collected in hedges close to vineyards in north eastern Italy. The majority of these plants were showing typical phytoplasma disease symptoms, such as yellowing, reddening, leaf roll and witches’ broom. Total nucleic acids were extracted from the leaf veins using the CTAB method (Angelini et al., 2001). In order to specifically amplify phytoplasma DNA, primer pairs P1/P7 followed by R16F2n/R16R2 were used in nested PCR.  Positive results were obtained in ten of the C. sanguinea and three of the S. nigra samples, although there was no correlation between the presence of phytoplasma and any specific symptoms.

Figure 1: Polyacrylamide gel electrophoresis of nested PCR products digested with enzymes TruI (left) and TaqI (right). Lanes 1 to 10 are C. sanguinea samples 1-10; Lanes 11-13 are S. nigra samples 1-3; Lane 14 is a stolbur reference strain. Lane 15 is a pBR322/HaeIII size marker (Sigma).

RFLP analysis in which the nested PCR products were digested with TruI and TaqI endonucleases was performed to identify the pathogen species. Differences between the samples were observed with both enzymes (Fig. 1). Two of the three S. nigra samples showed the same profiles as the stolbur reference isolate, revealing infections from a 16SrXII-A phytoplasma. However, all the other samples showed a different common pattern. One of these products was sequenced (GenBank Acc. No. EU127478) and BLAST analysis showed that the sequence matched 16SrXII-E phytoplasmas (EF015582 and DQ086423), with 100 and 99.9% identity respectively. This is a recently-described phytoplasma subgroup (Valiunas et al., 2006), members of which have only been isolated previously from strawberry plants in Lithuania and from Cordyline in the UK.

This is the first confirmed report of a 16SrXII-E infection in C. sanguinea and S. nigra plants. Samples of C. sanguinea collected in southern France had been found previously to be infected with a stolbur-like phytoplasma (Jarausch et al., 2001), but the pathogen subgroup had not been identified. This is also the first identification of a 16SrXII-A phytoplasma in S. nigra; yellows on this species had been observed previously (Marcone, 2002), but the aetiological agents had not been identified here either.

References

Angelini E, Clair D, Borgo M, Bertaccini A, Boudon-Padieu E, 2001. Flavescence dorée in France and Italy - occurrence of closely related phytoplasma isolates and their near relationships to Palatinate grapevine yellows and an alder yellows phytoplasma. Vitis 40, 79–86.

Jarausch W, Jarausch-Wehrheim B, Danet JL, Broquaire JM, Dosba F, Saillard C, Garnier M, 2001. Detection and identification of European stone fruit yellows and other phytoplasmas in wild plants in the surroundings of apricot chlorotic leaf roll-affected orchards in southern France. European Journal of Plant Pathology 107, 209-217.

Marcone C, 2002. Phytoplasma diseases of forest trees, landscape trees and shrubs in Europe. Petria 12, 381-386.

Valiunas D, Staniulis J, Davis RE, 2006. ‘Candidatus Phytoplasma fragariae’, a novel phytoplasma taxon discovered in yellows diseased strawberry, Fragaria x ananassa. International Journal of Systematic and Evolutionary Microbiology 56, 277-281.