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 wild plants (reservoirs). This study assessed the genetic variability of putative effector-coding genes (hp) in FDp strains collected in Northern Italy from symptomatic grapevines and additional plant hosts, and assigned to seven map genotypes [Epidemic: 16SrV-D (M54), 16SrV-C (M38, M50, M51); non-epidemic: 16SrV-C (M39, M43, M78)]. Based on the reference genome of strain CH (M54), 20 hp loci were selected and analyzed through an MLST approach. Sixteen loci were amplified in at least one genotype (hp13 and hp14 only in M54), while four loci (hp2, hp3, hp5, hp20) were not amplified. Sequence comparisons showed complete conservation of all amplified hp genes within M54 strains, regardless of host origin, whereas 16SrV-C genotypes displayed different divergence levels. In particular, genotype M50 showed the highest variability in hp gene sequences, including also intra-genotype variation. Loci hp1, hp8, hp12, and hp18 were the most variable, suggesting divergent strain-specific evolution and putative association with different biological behaviors. By comparing sequence variation among FDp strains infecting different hosts, this study provides new insights into the genetic diversity of putative virulence factors in FDp and contributes to a better understanding of the molecular basis underlying FD epidemiology.