Genetic diversity in putative effector genes among FDp and FDp-related strains from northern Italy

Flavescence dorée (FD) is the most important leafhopper-transmitted grapevine disease associated with a quarantine pathogen (FD phytoplasma, FDp). Recent studies demonstrated the presence of many FDp genotypes that differ in their epidemiological cycle, suggesting genotype-specific biological behaviour (Malembic-Maher et al., 2020; Rigamonti et al., 2023; Rizzoli et al., 2021). The recent availability of full FDp genome (Deboneville et al., 2022) can allow a deeper understanding of the dynamics that regulate this devastating disease, considering that, to date, phytoplasmas cannot be isolated in a pure culture in vitro, so they can be studied only at molecular level. The knowledge of genetic variability of putative effector genes in different FDp strains and how this variability can influence the disease development and severity will be useful to develop more targeted management strategies in agroecosystems. This study reports first results obtained by molecular typing of different FDp strains, identified in North Italy, based on sequence analyses of putative effector genes. MATERIALS AND METHODS Twenty-six 16SrV phytoplasma strains, previously identified in grapevine and other plant hosts in northern Italy, have been selected: 23 FDp strains (14 M54, 5 M51, 4 M50), and three FDp-related strains (2 M43, 1 M39) (Table 1). According to the FDp genome annotation by Deboneville et al. (2022), two putative effector genes (hp1, locus tag M6G77_00200; hp4, locus tag M6G77_02130), and a gene encoding the Bax inhibitor 1 (baxI, locus tags M6G77_00035 and M6G77_00935) have been selected for molecular typing. Primer pairs for each gene have been designed for direct and nested-PCR amplification carried out as follows: 94°C for 5 minutes followed by 35 cycles at 94°C for 1 min, 50°C (52°C in nested-PCR) for 1 min, 72°C for 2 min, and final elongation at 72°C for 7 minutes. PCR products have been sequenced in both directions. Nucleotide sequences and in-silico translated proteins have been compared to identify mutations within the analyzed FDp strains. RESULTS AND DISCUSSION Most of the 16SrV phytoplasma strains were positive to baxI gene amplification (25 out of 26), while 20 strains out of 22 and 18 out of 22 were positive to hp1 and hp4 amplification, respectively (Table 1). FDp and FDp-related strains belonging to the same map-genotype share identical baxI, hp1, and hp4 nucleotide gene sequences, while several SNPs (11 in baxI, 18 in hp1, 9 in hp4) were found distinguishing strains of distinct map-genotypes. Most of such SNPs (10 in baxI, 11 in hp1, 5 in hp4) were non-synonymous mutations, determining amino acidic variation in the in-silico translated proteins (Figure 1). None of the SNPs produced stop-codon interfering with the protein functionality. Differences observed in the putative effector genes and in-silico translated proteins, distinguishing FDp map-genotypes, could influence their interaction with hosts and determine their specific ecological niches. Further studies will be conducted to evaluate the genetic diversity of FDp and FDp-related map-genotypes in other putative effector genes, and to investigate and clarify the functional role of these putative effectors.