In vitro screening for resistance to apple proliferation in Malus species

A screening system for apple proliferation resistance was developed, based on in vitro graft-inoculation with the causal agent 'Candidatus Phytoplasma mali'. For this, in vitro cultures of the field-resistant apomictic genotypes Malus sieboldii, H0909, D2212 and the susceptible Malus x domestica genotypes Golden Delicious and rootstock M9 were established, as well as in vitro cultures of Rubinette and Golden Delicious infected with 'Ca. P. mali' strains PM4 and PM6, respectively. Healthy in vitro shoots were inoculated by micrografting with infected shoots used as graft tip. After 6 weeks graft contact no significant differences for graft quality were observed between healthy and infected grafts. Mortality of grafts and transmission rates were not significantly different among the different genotypes. The phytoplasma concentration in inoculated shoots was determined at different times post-inoculation (p.i.) by quantitative real-time PCR. Infected M. sieboldii and D2212 had lower phytoplasma concentration than the susceptible controls and showed no symptoms. H0909 showed an intermediate behaviour exhibiting lower phytoplasma concentrations with strain PM4 but growth was affected. The dynamics of phytoplasma concentration reached a maximum at 6-8 months p.i. for all genotypes but the values for 3-5 and 10-12 months p.i. were similar. The resistance of M. sieboldii and D2212 was confirmed in vitro. A significant difference in phytoplasma concentration was observed between strains PM4 and PM6.