Bois noir is a disease associated with the presence of phytoplasma ‘Candidatus Phytoplasma solani’ belonging to the Stolbur group (subgroup 16SrXII-A), which has a heavy economic impact on grapevines. This study focused on the changes induced by phytoplasma in terms of the profile and amount of secondary metabolites synthesized in the phenylpropanoid pathway in leaves of Vitis vinifera L. red-berried cultivar Sangiovese. Metabolic alterations were assessed according to the disease progression through measurements of soluble sugars, chlorophyll, and phenolic compounds produced by plant hosts, in response to disease on symptomatic and asymptomatic Bois noir-positive plants. Significant differences were revealed in the amount of soluble sugars, chlorophyll, and accumulation/reduction of some compounds synthesized in the phenylpropanoid pathway of Bois noir-positive and negative grapevine leaves. Our results showed a marked increase in phenolic and flavonoid production and a parallel decrease in lignin content in Bois noir-positive compared to negative leaves. Interestingly, some parameters (chlorophyll a, soluble sugars, total phenolic or flavonoids content, proanthocyanidins, quercetin) differed between Bois noir-positive and negative leaves regardless of symptoms, indicating measurable biochemical changes in asymptomatic leaves. Our grapevine cultivar Sangiovese results highlighted an extensive modulation of the phenylpropanoid biosynthetic pathway as a defense mechanism activated by the host plant in response to Bois noir disease.

Bois noir (BN) is the most widespread disease of the grapevine yellows complex in the Euro-Mediterranean area. BN is caused by ‘Candidatus Phytoplasma solani’ (BNp), transmitted from herbaceous plants to grapevine by polyphagous insect vectors. In this study, genetic diversity among BNp strains and their prevalence and possible association with grapevine symptom severity were investigated in a Sangiovese clone organic vineyard in the Chianti Classico area (Tuscany). Field surveys over 2 years revealed a range of symptom severity on grapevine and an increase of BN incidence. A TaqMan allelic discrimination assay detected only tufB type b among BNp strains, suggesting the prevalence of the bindweed-related ecology. Nucleotide sequence analyses of vmp1 and stamp genes identified 12 vmp1 and 16 stamp sequence variants, showing an overall positive selection for such genes. The prevalent genotype was Vm43/St10, reported for the first time in this study and closely related to strains identified only in the French Eastern Pyrenees. BNp strains identified in the examined vineyard and mostly grouped in separate bindweed-related phylogenetic clusters showed statistically significant differences in their distribution in grapevines exhibiting distinct symptom severity. These results suggest the possible occurrence of a range of virulence within BNp strain populations in the Chianti Classico area.

Bois noir (BN), associated with ‘Candidatus Phytoplasma solani’ (CaPsol), is the most widespread disease of the grapevine yellows complex worldwide. In this work, BN epidemiology was investigated in a case study vineyard where an unusual CaPsol strain, previously detected only in other host plants, was found to be prevalent in grapevine. Experimental activities included: symptom observation; sampling of symptomatic vines, Auchenorrhyncha specimens, and weeds; molecular detection and typing of CaPsol strains; statistical analyses for determining possible relationships between CaPsol relative concentration, strain type, and symptom severity. Among insects, Reptalus quinquecostatus was the most abundant and was found to be highly infected by CaPsol, while Hyalesthes obsoletus, the main CaPsol vector, was not caught. Moreover, R. quinquecostatus harbored CaPsol strains carrying uniquely the stamp sequence variant St10, also identified as prevalent in vines and in the majority of weeds, and all the secY variants identified in the vineyard. Statistical analyses revealed that CaPsol strains carrying the St10 variant are not associated with severe symptoms, suggesting their possible moderate virulence. Based on such evidence, a new BN epidemiological pattern related to these CaPsol strains and involving grapevine, R. quinquecostatus, and/or weeds is proposed. Furthermore, the possible presence of other players (vectors and weeds) involved in CaPsol transmission to grapevines was highlighted.

“Bois noir” disease associated with ‘Candidatus Phytoplasma solani’ seriously compromises the production and survival of grapevines (Vitis vinifera L.) in Europe. Understanding the plant response to phytoplasmas should help to improve disease control strategies. Using a combined metabolomic and transcriptomic analysis, this work, therefore, investigated the phytoplasma–grapevine interaction in red cultivar Sangiovese in a vineyard over four seasonal growth stages (from late spring to late summer), comparing leaves from healthy and infected grapevines (symptomatic and symptomless). We found an accumulation of both conjugate and free salicylic acids (SAs) in the leaves of ‘Ca. P. solani’-positive plants from early stages of infection, when plants are still asymptomatic. A strong accumulation of gentisic acid (GA) associated with symptoms progression was found for the first time. A detailed analysis of phenylpropanoids revealed a significant accumulation of hydroxycinnamic acids, flavonols, flavan 3-ols, and anthocyanin cyanidin 3-O-glucoside, which are extensively studied due to their involvement in the plant response to various pathogens. Metabolomic data corroborated by gene expression analysis indicated that phenylpropanoid biosynthetic and salicylic acid-responsive genes were upregulated in ‘Ca. P. solani-positive plants compared to -negative ones during the observed period.

Grapevine Bois noir (BN) is associated with infection by “Candidatus Phytoplasma solani” (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of tuf-type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene hlyC, which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of tufB gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu (tufB gene) and hemolysin III-like protein (hlyC gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of tuf-type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards.