Murolo, Sergio

‘Candidatus Phytoplasma solani’ is the causal agent of the Bois noir (BN), affecting grapevine worldwide. The complex epidemiology of BN, which involves multiple ‘Ca. P. solani’ host plants and insect vectors, as well as the occurrence of recovery (loss of symptoms on grapevine canopy), makes disease investigations and containment in vineyards difficult. To achieve early detection of ‘Ca. P. solani’, a droplet digital PCR (ddPCR)-based approach and quantitative (q)PCR assay were compared, testing specific primers based on the elongation factor Tu (tuf) gene using SYBR Green chemistry. The regression curve analysis of the ddPCR assay showed good linearity. Compared with the qPCR method, the sensitivity of ddPCR improved about 10-fold. The analysis of grapevine roots spiked with serial dilutions of ‘Ca P. solani’. PCR tuf fragments showed that qPCR was inhibited, while ddPCR was not affected. Testing 66 grapevine samples from 50 grapevine plants, the ddPCR provided superior diagnostic performance compared to qPCR in roots of symptomatic plants (75% detected by ddPCR, 41.6% by qPCR), roots of recovered plants (58.8% detected by ddPCR, 25% by qPCR), and asymptomatic leaf tissues from recovered plants (75% detected by ddPCR, 25% by qPCR). The ddPCR analysis allowed us to detect ‘Ca. P. solani’ on 40% of leaf samples from recovered plants and 20% of roots from asymptomatic plants. No differences among ddPCR and qPCR were found in detecting phytoplasma on symptomatic leaf samples. The ddPCR assay allowed the absolute quantification of ‘Ca. P. solani’ in complex matrices, such as roots, and when low titer of phytoplasma is present.

Grapevine Bois noir (BN) is associated with ‘Candidatus Phytoplasma solani’. It has been recorded in vineyards throughout Europe as well as in different countries in Asia, where it now constitutes a threat to Iranian viticulture. BN is strictly dependent on ‘Ca. P. solani’ strains, wild host plants, and insect vectors. The molecular typing of ‘Ca. P. solani’, based on the nonribosomal gene tuf and the two hypervariable markers vmp1 and stamp, is valuable for the reconstruction and clarification of the pathways of BN spread. In this study, an RFLP analysis was performed on the vmp1 gene, and a single-nucleotide polymorphism analysis confirmed new vmp types in ‘Ca. P. solani’. A stamp gene phylogenetic analysis allowed us to distinguish between the new genotype infections in the grapevines and the ‘weeds’ Convolvulus arvensis and Erigeron bonariensis in Iranian vineyards, highlighting the close genetic relatedness of the strains of ‘Ca. P. solani’ found in Iran and Azerbaijan. The most common genotype in the grapevines was tuf b/V24/stamp III, which was associated with C. arvensis. This information contributes toward the identification of further routes of introduction of ‘Ca. P. solani’ in Iran to sustain the control measures for the management of BN.

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.

Bois noir is caused by ‘Candidatus Phytoplasma solani’, and it is one of the most important and widespread diseases in the Euro-Mediterranean region. There are complex interactions between phytoplasma and grapevines, weeds, and vectors. These ecological relationships can be tracked according to molecular epidemiology. The aims of the 2-year study (2014–2015) were to describe incidence and spatial distribution of Bois noir in a vineyard with three grapevine varieties in Sicily, and to identify the molecular types of the tuf and vmp1 genes in these naturally infected grapevines, according to the potential reservoir plants and vectors. Disease incidence in 2015 was significantly higher in ‘Chardonnay’ (up to 35%) than for ‘Nero d’Avola’ and ‘Pinot noir’ (<5%). All grapevine, weed, and insect samples were infected by ‘Ca. P. solani’ tuf-type b. Most of the collected insects were strictly related to Vitis spp. and belonged to Neoaliturus fenestratus, Empoasca spp., and Zygina rhamni. The characterization of the vmp1 gene revealed six different vmp types in grapevines (V1, V4, V9, V11, V12, V24), three in weeds (V4, V9, V11), and four in insects (V4, V9, V11, V24). Notably, V4, V9, appear both in hosts and vectors, with V9 predominant. Virtual restriction fragment length polymorphism (RFLP) analysis based on the nucleotide sequences supported the data of the conventional RFLP. Connections between the molecular data recorded in the vineyard ecosystems and the application of innovative tools based on the geostatistical analysis will contribute to further clarification of the specific ecological and epidemiological aspects of ‘Ca. P. solani’ in Sicily.

Grapevine Bois noir (BN) is caused by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) and is one of the most important phytoplasma diseases in the Euro-Mediterranean viticultural areas. The epidemiology of BN can include grapevine as a plant host and is usually transmitted via sap-sucking insects that inhabit herbaceous host plants. Tracking the spread of ‘Ca. P. solani’ strains is of great help for the identification of plant reservoirs and insect vectors involved in local BN outbreaks. The molecular epidemiology of ‘Ca. P. solani’ is primarily based on sequence analysis of the tuf housekeeping gene (which encodes elongation factor Tu). In this study, molecular typing of tuf, through restriction fragment length polymorphism and sequencing, was carried out on grapevine samples from Iranian vineyards. According to the molecular characterization, three molecular types—tuf b1, tuf b5 and tuf b6—were found, with tuf b1 being the most prominent. These data provide further knowledge of tuf gene diversity and question the ecological role of such “minor” tuf types in Iranian vineyards, which have been detected only in grapevines.

Grapevine (Vitis vinifera L.) is one of the most important horticultural crops in Iran, with >200,000 ha of cultivated area. Recently, outbreaks of the grapevine yellows Bois noir that is associated with phytoplasma strains related to ‘Candidatus Phytoplasma solani’ were recorded in several Iranian regions. This has resulted in severe economic losses. We carried out a survey in 2015, followed by collection of leaf samples from symptomatic grapevines and weeds. Because no information is available on the molecular epidemiology of ‘Ca. P. solani’ in Iran, multiple gene analyses were carried out here according to molecular characterization of the tuf and vmp1 genes. From the molecular characterization, all of the samples (i.e., grapevines, weeds) were infected with tuf b type. Detailed molecular characterization of the vmp1 gene (i.e., PCR–restriction fragment length polymorphism, sequence analysis) defined five molecular types: V1, V4, V10, V15, and V20. The abundance of Convolvulus arvensis in vineyards and detection of the same ‘Ca. P. solani’ molecular types in grapevines and weeds suggest that C. arvensis has a major role in Bois noir epidemiology of Iranian vineyards. Therefore, control strategies should be developed to manage these host plants to reduce inoculum sources of the phytoplasma in vineyards.

Abstract‘Candidatus Phytoplasma solani’ is the causal agent of Bois noir (BN) in grapevine (Vitis vinifera). It is usually detected in leaves, where typical disease symptoms are seen. However, little information is available on the presence of this phytoplasma in grapevine roots. Here, we investigated ‘Ca. P. solani’ in roots collected from 28 symptomatic, 27 recovered and eight asymptomatic grapevine plants. Protocols based on high-resolution melting (HRM) combined with real-time quantitative PCR (qPCR-HRM) and nested-qPCR-HRM were developed to identify ‘Ca. P. solani’ tuf-type variants with single nucleotide polymorphisms. In all, 21.4% of roots from symptomatic plants were positive to ‘Ca. P. solani’ using qPCR-HRM, and 60.7% with nested-qPCR HRM. Also, 7.4% of roots from recovered plants were positive using qPCR-HRM, which reached 44.4% using nested-qPCR HRM. These analyses identified tuf-type b1 on 88.2% of the positive samples from symptomatic grapevines, and 66.6% from recovered grapevines, with all other samples identified as tuf-type a. This study reports the presence of ‘Ca. P. solani’ in the roots of both symptomatic and recovered grapevines. These qPCR-HRM and nested-qPCR-HRM protocols can be applied to increase the sensitivity of detection of, and to simplify and speed up the screening for, ‘Ca. P. solani’ tuf-types.