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Phytoplasmas ('Candidatus Phytoplasma') are plant-pathogenic bacteria that colonize the phloem of their host plants and can cause diseases in more than 700 plant species world-wide, including many economically important crops, ornamentals, and forest trees. They are spread by phloem-feeding insect vectors, vegetative propagation (grafting or production of cuttings), or plant parasitic dodder species of the genus Cuscuta.
In Rubus species, phytoplasmas are associated with a disease referred to as Rubus stunt. Symptoms include stunting, witches' broom, small leaves, short internodes, enlarged sepals, phyllody, flower proliferation, and fruit malformations. As Rubus species are usually propagated vegetatively and the period of latency can be up to one year, the commercial production of planting material in plant nurseries can play a major role in the spread of Rubus stunt in addition to its only known insect vector, the leafhopper Macropsis fuscula. Furthermore, phytoplasmas usually occur in Rubus plants at concentrations too low for regular PCR to detect their DNA in plants and nested PCR is too time consuming for routine screening and prone to carry-over contamination. Hence, a fast, sensitive, and reliable molecular detection method for phytoplasmas in Rubus species is of major importance to stop the spread of this disease. Therefore, a multiplex TaqMan qPCR assay for the detection of Rubus stunt phytoplasmas was developed in this thesis, which provides an efficient tool for the screening of Rubus mother plants.
This qPCR assay was subsequently used in a screening of putative insect vectors of Rubus stunt phytoplasmas in raspberry plantations in southern and northern Germany. A total of 4868 hemipteran insects were sorted, identified to family, genus, or species level and 597 DNA extracts, including pooled samples representing all identified insects, sampling locations, and sampling dates, were analyzed for phytoplasma DNA. With only seven sampled individuals of M. fuscula the occurrence of the only known vector for Rubus stunt phytoplasmas was low. DNA extracts from 18 samples were positive for phytoplasma DNA, among them species from the genera Euscelidius, Macrosteles, Euscelis, Anaceratagallia, and Psammotettix.
Furthermore, the susceptibility of four raspberry cultivars to Rubus stunt after artificial graft inoculation was investigated. While graft inoculation was verified to be a practical tool for artificial phytoplasma inoculation in raspberries, assessing the disease severity of Rubus stunt proved to be difficult. All four raspberry cultivars used in this experiment ('Autumn Bliss', 'Glen Ample', 'Polka', and 'Tulameen') were successfully colonized by phytoplasmas.
The efficacy of heat therapy with subsequent tissue culture to eliminate phytoplasmas from infected raspberry and blackberry plants was also evaluated. All previously infected raspberry and blackberry plants were tested negative for the presence of phytoplasma DNA after this procedure. Therefore, this method is suitable as an important step during the production of healthy mother plants and nuclear stock material in order to secure the production of healthy planting material.
In conclusion, a fast and reliable molecular detection assay for routine diagnostics of phytoplasmas in Rubus species was developed and applied in experiments investigating putative insect vectors of Rubus stunt phytoplasmas, the susceptibility of different raspberry cultivars, and the efficacy of heat therapy coupled with tissue culture. The results of these trials can now be incorporated into disease management strategies for Rubus stunt.