Comparison of different techniques for inoculation of "Candidatus Phytoplasma mali" on apple and periwinkle in biological indexing procedure.

As phytoplasmas are non cultivable micro-organisms, the research on phytoplasmal diseases can only be achieved with infected hosts. Biological indexing (by grafting) is the simplest detection method for phytoplasmal diseases. We tested four different grafting techniques for inoculation of apple trees or periwinkles in greenhouse, including whip graft, bark graft, budding and chip-budding. All techniques were tested on apple trees (six trees per phytoplasma isolates) in insect-proof greenhouse. The whip and bark grafting were not feasible for periwinkle plants, because of fineness and fragility of their tissues: only the chip-budding was performed (four plants per isolate). In apple trees, the best and soonest positive results were obtained by chip and bark grafting. Except for seven transplants not-grown after grafting, 100% efficiency of inoculation was obtained by both methods. Nevertheless, the transmission of phytoplasma from transplant not-grown to rootstock was sometimes recorded (28.6%). The earliest phytoplasma symptoms after whip or bark grafting appeared after 3 months. Symptoms were obtained much later with budding and chip-budding. In case of periwinkles, infected apple and periwinkle materials were used as inoculum sources. Transmission of phytoplasma from periwinkle to periwinkle was successfully carried out by chip-budding grafting. The symptoms were observed during the second month after inoculation. The transmission of phytoplasma from infected apple material to periwinkle (by chip-budding) was achieved for 60 % of the tested samples. Moreover, the latency period before symptom observation was longer. Finally, we perceived the apple trees are more convenient and rapid than periwinkle plants for biological indexing of apple materials.