Long-term maintenance of nonculturable apple-proliferation phytoplasmas in their micropropagated natural host plant

Phytoplasmas associated with apple proliferation (AP) disease of apple trees have been maintained in their micropropagated natural host plant Malus pumila since 1985. Different isolates of these nonculturable plant pathogens could thus be studied in vitro. Amplification of a pathogen-specific DNA fragment by polymerase chain reaction (PCR) confirmed the presence of AP phytoplasmas in the diseased plants even after 10 years of in-vitro propagation. Restriction fragment length polymorphism analysis of the amplified chromosomal DNA fragments revealed no genetic difference between the AP phytoplasma isolates. Growth parameters, symptom expression and phytoplasma concentration were examined to compare the in-vitro behaviour of four different AP phytoplasma isolates and to compare different subculture conditions. A comparison of these data obtained after 2 or 8 years of micropropagation revealed no essential differences. Eight years after culture initiation, diseased shoots still exhibited typical symptoms like witches' broom, small leaves with large stipules and stunted growth. The use of phytoplasma-diseased micropropagated plants to establish a 'type culture collection' of these otherwise nonculturable plant pathogens is discussed.