Multilocus sequence typing of phytoplasma strains associated with "bois noir" in Italian vineyards

Background and objectives Bois noir (BN) is an important grapevine yellows (GY) that is widely spread in Europe and in the Mediterranean basin. BN is attributed to infection of Vitis vinifera by 'Candidatus Phytoplasma solani' strains that are transmitted plant-to-plant mainly by the cixiid Hyalesthes obsoletus Signoret (Quaglino et al., 2013). Complex BN epidemiology related to the numerous weed plants hosting BN phytoplasmas (BNp) within and around vineyards, and to the possible presence of additional vector(s) has increased the necessity to study the genetic diversity among BNp populations in diverse ecological niches. Recent studies, based on molecular characterization of more (16S rRNA, tuf, secY) or less (vmp, stamp) conserved gene nucleotide sequences, evidenced an unexpectedly high genetic diversity among BNp strains from European Countries (Foissac et al., 2013). In the present work, multilocus sequence typing (MLST) analyses including 16S rRNA, tuf, hlyC, cbiQ-glyA, trxA-truB, and rplS-tyrS-csdB were carried out for investigating the genetic diversity among BNp populations from distinct regions of Italy. Materials and Methods BNp strains identified in 108 grapevine plants collected from 2008 to 2012 in vineyards located in Lombardia (north-western Italy), Veneto (north-eastern Italy), Marche and Abruzzi (central Italy), and Sicily (southern Italy) regions were characterized by Polymerase Chain Reaction (PCR)-based amplifications of the genes 16S rRNA, tuf, hlyC, cbiQ-glyA, trxA-truB, and rplS-tyrS-csdB, followed by Restriction Fragment Length Polymorphism (RFLP) analyses. Reaction conditions were as previously described (Quaglino et al., 2009). PCR products and RFLP patterns were visualized on 1% and 3% agarose gels, respectively, under a UV transilluminator. Results and Discussion RFLP analyses performed on 16S rDNA amplicons revealed that all BNp strains analyzed in the present work belong to taxonomic subgroup 16SrXII-A. Based on tuf gene characterization, BNp strains were divided in two types, BNp-type I (formerly called VK-I or tuf type A) and BNp-type II (formerly called VK-II or tuf type B). BNp-type I was prevalent (65 strains) in the examined Italian vineyards, but prevalence of the two BNp-types differed depending on the region studied. Thus, BNp-type I was prevalent in Veneto; type II in Marche/Abruzzi and in Sicily. In Lombardia, BNp-types I and II were almost identical in regional prevalence. Based on hlyC RFLP analyses, two distinct restriction patterns were found, distinguishing the BNp strains into two groups consistent with BNp-types. Based on cbiQ-glyA, trxA-truB, and rplS-tyrS-csdB RFLP analyses, it was possible to identify two distinct restriction profiles for each of those genomic segments, distinguishing the BNp strains into groups that were not consistent with those defined by tuf/hlyC gene RFLP patterns. Considering the cbiQ-glyA, trxA-truB, and rplS-tyrS-csdB RFLP profiles of each BNp strain as unique collective RFLP pattern, it was possible to evidence the presence of eight collective patterns: four associated with BNp-type I and II; three unique for BNp-type II; and one unique for BNp-type I. In detail, five and seven collective patterns were identified among BNp-type I and II strains, respectively, showing a possibly higher genetic variability among BNp-type II. Such evidence reinforces previous results obtained by vmp1, stamp, and groEL gene sequence analyses (Murolo et al., 2010; Foissac et al., 2013; Mitrović et al., 2013). On the basis of overall RFLP collective patterns, MLST analyses including all the analyzed genes evidenced the presence of 12 BNp lineages (BNp1 to BNp12) among BNp phytoplasma populations analyzed in the present work. Prevalent lineages were BNp1 (24 strains), BNp4 (20 strains) and BNp12 (15 strains), but in each examined region the lineage prevalence was found different. In detail, BNp1 and BNp4 were prevalent in northern Italy, BNp12 in central Italy, and BNp6 in southern Italy. Interestingly, no BNp lineage was found in every region studied, suggesting a possible influence of region-specific weed host plants and/or insect vector(s) in selecting BNp strain lineages and determining regional BNp population composition. Molecular markers identified in the present study will be employed for investigating more accurately the presence of BNp lineages in grapevine, weed host plants and insect vector(s) in diverse ecological niches in order to clarify the BN epidemiology.