Molecular differentiation of severe and mild strains of ‘Candidatus Phytoplasma mali’ and evidence that their interaction in multiply infected trees determines disease severity

Previous work has shown that multiple infections of apple trees by distinctly different strains of ‘Candidatus Phytoplasma mali’ are widespread. In the current study, pathological data of infected trees with single or multiple phytoplasma were analyzed and compared with molecular data based on a hflB gene of the infecting phytoplasmas. Single-strand conformation polymorphism and sequence analysis of a variable hflB gene fragment revealed that mild and severe strains can be distinguished by their SSCP profiles and their phylogenetic clustering. Analysis of cloned sequences from 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. Our 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 may occur that drastically alter virulence of multiple-strain accessions.