Multilocus Genotyping Reveals New Molecular Markers for Differentiating Distinct Genetic Lineages among “Candidatus Phytoplasma Solani” Strains Associated with Grapevine Bois Noir

Abstract

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.

Epidemiological role of novel and already known ‘Ca. P. solani’ cixiid vectors in rubbery taproot disease of sugar beet in Serbia

“…CaPsol strains belonging to the tuf-b epidemiology, reported previously 4 and in this study as involved in RTD, along with strains PO (St10) and RQ161 (St46) detected in R. quinquecostatus in Italy and France 16,29 , were subjected to M-J analysis. The tuf gene was amplified by combining two PCR procedures, depending on the amplification efficacy: a nested protocol with primer pairs fTuf1/ rTuf1 and fTufAY/rTufAY 21,48 and a semi-nested protocol with fusAF1/tufBR1 and fusAF2/tufBR1 primers 49 . Further identification of the tuf type/genotype of the analysed CaPsol samples was done as previously described 4 .…”

Multilocus Genotyping of ‘Candidatus Phytoplasma solani’ Associated with Rubbery Taproot Disease of Sugar Beet in the Pannonian Plain

“…In order to amplify the tuf gene, the Tuf1f/r primer pair was followed by fTufAy/rTufAy in nested PCR assays [ 42 ]. For those samples in which no, or very weak, amplification was obtained, an alternative semi-nested protocol was used for amplifying the tuf gene, applying fusAF1/tufBR1 primers, followed by fusAF2/tufBR1 [ 16 ]. Nested amplicons of both PCR systems were separated and visualised as described above.…”

“…While the ecological and epidemiological significance of the tuf-d type is still to be evaluated, recent studies in Asia (Iran) have boosted ‘ Ca . P. solani’ strains genotypisation based on this particular gene [ 15 , 16 ]. Molecular characterisation on highly variable genes such as stamp and vmp1 (variable membrane protein), has already been used, not only to access genetic variability, but also to give an insight into the complex epidemiological cycles of these plant pathogenic microorganisms that spread through natural and cultivated vegetation [ 12 , 17 , 18 , 19 ].…”

Molecular detection and multilocus sequence analysis of Candidatus Phytoplasma solani-related strains infecting potato and sugar beet plants in Southern Germany

“…Since the routinely used 16SrDNA sequences often is not su cient for strain discrimination and epidemiological investigations (Ray, 2013), additional molecular marker such as tuf (translation elongation factor Tu), secY (translocation protein SecY), vmp1 (variable membrane protein) and stamp (stolbur phytoplasma antigenic membrane protein) genes have been used to distinguish various strains of 'Ca. P. solani' (Quaglino et al, 2013, Fabre et al, 2011, Passera et al, 2020. Using this technique for identi cation of 'Ca.…”