Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-6921 Dossenheim, Germany
For competitive polymerase chain reaction (PCR), an internal standard DNA template was developed that consisted of a highly conserved, internally deleted 16S rDNA fragment of an aster yellows phytoplasma. The internal standard was calibrated using a quantified culture of Acholeplasma laidlawii. Serial dilutions of the internal standard and fixed amounts of target templates from infected plants were coamplified with the same primers, and the products obtained were quantified using an enzyme-linked immunosorbent assay procedure. Analysis of the data revealed that the phytoplasma concentration in the plants examined differed by a factor of about 4 × 106. Phytoplasma concentrations of 2.2 × 108 to 1.5 × 109 cells per g of tissue were identified in periwinkles infected with various phytoplasmas. High to moderate concentrations were detected in Malus domestica (apple) genotypes infected with the apple proliferation phytoplasma, Alnus glutinosa (alder) genotypes infected with the alder yellows phytoplasma, and most aster yellows-infected Populus (poplar) genotypes examined. Very low phytoplasma concentrations, ranging from 370 to 34,000 cells per g of tissue, were identified in proliferation-diseased apple trees on resistant rootstocks 4551 and 4608, yellows-diseased Quercus robur (oak) trees, and Carpinus betulus (hornbeam) trees. Such low concentrations, which corresponded to about 4 to 340 cells in the reaction mixture, could only be detected and quantified by nested PCR.