Molecular evidence for mixed phytoplasma infection in lily plants

During inspection of lily plantations grown in Poland from imported bulbs of five different hybrids, plants with severe chlorosis, leaf necrosis and malformation, dropping and abortion of flower buds were observed. Most of the plants were infected with lily symptomless virus (LSV) and some of them also with cucumber mosaic virus (CMV). In order to verify phytoplasma presence, bulb scales and periwinkles infected by grafting were tested by nested PCR followed by RFLP analyses. To clarify phytoplasma identity, several primer pairs, amplifying 16S ribosomal DNA, were employed; RFLP analyses on the amplified fragments allowed the identification of a mixed phytoplasma infection in the majority of lily and periwinkle samples. Identified phytoplasmas belong to groups 16SrI-B and 16SrXII-A. Further PCR experiments were carried out on the gene coding for ribosomal protein L22 (primers Pr1/Pr2), on a sequence coding for a probable protein releasing factor (primers BB88F1/R1) and on the tuf gene coding for the elongation factor EF-tu. In the majority of samples no reliable results were obtained with these primers and only the positive controls were amplified. The symptoms expressed by infected lily plants suggest an interaction between the two phytoplasmas detected. Moreover the results obtained indicated that the phytoplasma concentration in the material employed must be not very high, probably in relationship to the environmental growing conditions, i.e. low temperature.