Identification and molecular characterization of multiple phytoplasma infection in Spartium junceum and Cytisus scoparius

Two genera of the Fabaceae family showed phytoplasmas symptoms in different locations in Europe i.e. Germany and Italy. Spartium junceum L. (Spanish broom) is a deciduous shrub with dark green, round stems and alternate leaves; inflorescences are terminal clusters of several bright yellow somewhat fragrant flowers. This ornamental shrub is frequently spontaneously growing especially in southern Italy where it is affected by spartium witches’ broom (SpaWB) disease, characterized by proliferation of axillary buds and stem fasciation. Two different phytoplasmas have been associated to SpaWB: ‘Candidatus Phytoplasma spartii’ (group 16SrX-D) and a phytoplasma belonging to elm yellows group (16SrV-C). Both were reported associated with SpaWB in Italy while only ‘Ca. P. spartii’ was reported in Spain. In the spring of 2011, typical SpaWB symptoms were observed in a plant up to 2 m tall growing in the city of Ercolano (Campania region, Italy). A similar symptomatology was observed in a group of shrubs of Cytisus scoparius (L) syn. Sarothamnus scoparius, better known as common broom or scotch broom growing in Dahlem botanical garden in Berlin (Germany). This is a perennial shrub native to western and central Europe, but it is considered invasive plant in areas such as North America and New Zealand. Symptomatic and asymptomatic samples were collected in both cases: five samples of C. scoparius and two of S. junceum were analysed for phytoplasma presence by nested-PCR assays employing primer pairs P1A/P7A followed by F1/B6 and R16F2n/R2, phytoplasma identification was achieved by RFLP analyses with Tru1I on the two latter amplicons. Further confirmation of phytoplasma identity was achieved by nested-PCR assays with primers specific for phytoplasma groups 16SrI, 16SrV and 16SrX. All symptomatic samples produced amplicons of the expected lengths and no product was amplified from asymptomatic plants and using 16SrV specific primers. Identification and classification of phytoplasmas allow to detect ‘Ca. P. spartii’ subgroup 16SrX-D and ‘Ca. P. asteris’ subgroup 16SrI-B in both genera. In some of the samples of C. scoparius also stolbur phytoplasmas were identified. Further phytoplasma characterization was carried out on tuf gene using a cocktail primers mix that was able to amplify phytoplasmas identified as ‘Ca. P. asteris’ in S. junceum and phytoplasmas showing two different Tru1I profiles in C. scoparius from Germany that are not present in any published RFLP profile on this gene. Direct amplicon sequencing is in progress in order to verify possible affiliation to ‘Ca. P. spartii’ group since the only available sequences of this gene on phytoplasmas are deposited in Qbank since they were obtained from the Qbol EU project.