Multigene Typing of Croatian ‘Candidatus Phytoplasma Mali’ Strains

Phytoplasmas ('<i>Candidatus</i> Phytoplasma') are intracellular pleomorphic plant pathogens belonging to the class Mollicutes. They colonize both plant hosts and insect vectors in their life cycle. Apple proliferation (AP) is one of the most important phytoplasmoses present in Europe, causing significant economic losses in apple production. The causal agent, '<i>Ca</i>. P. mali', was identified in apple and <i>Cacopsylla picta</i> samples using both real-time PCR and nested PCR based on the amplification of 16S rDNA. The objective of this study was to gain deeper insights into the epidemiology of apple proliferation in Croatia. Variability of genetic markers other than <i>16S rRNA</i> was used for characterization of strains. Four molecular markers differing in level of conservation, <i>aceF</i>, <i>pnp</i>, <i>imp</i>, and <i>secY</i>, were selected in line with previously typed fruit tree phytoplasmas. New genotypes were discerned for each genetic marker, and 20 different sequence types were revealed in the Croatian strains of '<i>Ca</i>. P. mali'. On the basis of this comprehensive analysis, the founder sequence type ST1 (A13-P10-S12-I21) can be proposed. This is the first extensive research and multigene typing performed on Croatian '<i>Ca</i>. P. mali' strains. Obtained results reveal considerable genetic diversity of epidemiological relevance limited to only two locations in north-western Croatia. Additionally, novel primers were constructed to amplify fragments larger than the entire coding region for all four genes in order to further expand the phytoplasma multi-locus sequence typing scheme.