HfIB Gene-Based Phytopathogenic Classification of 'Candidatus Phytoplasma mali' Strains and Evidence that Strain Composition Determines Virulence in Multiply Infected Apple Trees

Analysis of pathological and molecular data of 'Candidatus Phytoplasma mali' accessions from 27 apple trees differing considerably in symptomatology was used to molecularly characterize and classify strains of the infecting apple proliferation phytoplasma. Single-strand conformation polymorphism and sequence analysis of a variable fragment of ATP00464-type hflB gene revealed that these sources consisted of single-strain and multiple-strain accessions that occurred in similar numbers. The latter group was composed of two to five distinct strains. Analysis of cloned sequences of mild and severe single-strain accessions resulted in two groups of reads that clustered, according to their virulence, distantly in the phylogram. Based on this data, the clustering patterns of multiple-strain accession sequences indicated that nearly all of them were composed of mild and severe strains. The distinct clustering of sequences representing mild and severe strains was associated with a range of molecular markers at the nucleotide and amino acid level. Data indicate that the virulence of multiple-strain accessions is determined by the ratio of the occurring mild and severe strains in that mild accessions were characterized by the predominance of sequences representing mild strains and vice versa. There is evidence that shifts in the population and other events may occur that drastically alter virulence of multiple-strain accessions.