Putative Effector Genes Reveal Hidden Genetic Diversity Beyond Conventional Typing Markers in Flavescence Dorée Phytoplasmas

Flavescence dorée (FD) is a severe grapevine disease associated with FD phytoplasmas (FDp), mainly transmitted by the insect vector Scaphoideus titanus and occurring also in symptomless reservoir plants. This study assessed the genetic variability of putative effector-coding genes (hp) in FDp and FDp-related strains from Northern Italy, assigned to epidemic (M54, M38, M50, M51) and non-epidemic (M39, M43, M78) map genotypes. Based on the reference genome of FDp strain CH, 20 hp loci were selected and analyzed through a multi-locus sequence typing approach. Sixteen loci were amplified in at least one genotype, while four loci failed to amplify under the conditions used. Sequence comparisons showed complete conservation of all amplified hp genes within M54 strains, whereas other genotypes displayed different divergence levels, with the greatest heterogeneity observed in the M50 genotype, including also intra-genotype variation. Several loci exhibited substantial strain-level variability, suggesting locus- and strain-dependent diversification. However, this variability was not consistently associated with biological or epidemiological traits of FDp strains, such as host plant or epidemic status, and its functional relevance therefore remains unresolved. Overall, these results provide a first characterization of sequence diversity in candidate virulence-related loci across FDp genetic backgrounds, revealing additional genetic diversity beyond conventional FDp typing markers and supporting further functional investigation of their possible role in host or vector interactions.