Strain differentiation of Candidatus Phytoplasma mali by SSCP and sequence analyses of the HFLB gene

Molecular analysis of the hflB gene ATP00464 of Candidatus Phytoplasma mali strains revealed sequence polymorphisms within this taxon. A 530 bp fragment from 42 accessions originating from apple proliferationinfected plants from Germany, Italy, and France was amplified with primers fHflB3-1/rHflB3. The variability of the amplimers was demonstrated by single-strand conformation polymorphism (SSCP) analysis that resulted in more than 20 different profiles. PCR fragments were sequenced to corroborate SSCP results. The nucleic acid homology within Ca. P. mali strains ranged from 94.2% to full identity, whereas the identity to the hflB gene of Candidatus Phytoplasma pyri and Candidatus Phytoplasma prunorum was at least 85.9% and 84.6% or higher, respectively. The homology on the protein level within Ca. P. mali was higher as a number of substitutions were silent mutations. The phylogenetic tree derived from the nucleic acid sequences revealed more than 20 different lineages and agreed with the SSCP typing. The molecular analysis of the hflB gene in combination with SSCP typing allowed high throughput analysis of samples and enabled differentiation of Ca. P. mali strains to a higher degree than published markers. The polymorphism of the hflB fragment predestine this gene as a molecular marker for tracking strains in epidemiological studies and characterizing, define and monitor inoculum strains used for resistance breeding projects.