Stamp Genes Research Articles

Identification of phytoplasmas associated with grapevine ‘bois noir’ and flavescence dorée in inter-row groundcover vegetation used for green manure in Franciacorta vineyards

‘Bois noir’ (BN) and flavescence dorèe (FD), the two main diseases of the grapevine yellows complex associated with genetically distinct phytoplasmas, have a complex epidemiology including multiple insect vectors and reservoir plants. This study investigated the presence of BN and FD phytoplasmas in nine groundcover plant species commonly utilized for inter-row vineyard green manure in Franciacorta (North Italy). The activities conducted in 2020 included monitoring and sampling groundcover plant species and symptomatic grapevines in September, and phytoplasma identification and typing by amplification and sequence analyses of stamp and map genes. Molecular analyses identified BN phytoplasma (strains carrying the stamp gene sequence variants St5, St19, St30) and FD phytoplasma (strains carrying the map gene sequence variant M54) in 72% and 28% of symptomatic grapevines, respectively. BN phytoplasma strains St5 and St30 were found also in Eruca sativa, Vicia sativa, and Polygonum fagopyrum. FD phytoplasma strain M54 was found also in Vicia faba, Trifolium incarnatum, and Polygonum fagopyrum. These results reinforced the evidence of the increasing range of BN and FD phytoplasma alternative plant hosts and suggested a criterium for the selection of the groundcover plant species utilized for green manure, excluding the ones putatively involved in BN and FD diffusion.

Promoter Region Analysis of STAMP1/STEAP2 Gene-Silencing of STAMP1/STEAP2 Gene Triggers P53 Upregulation

Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer mortality in men in the Western World. In the initial stages, prostate cancer is dependent on androgens for growth which is the basis for androgen ablation therapy. The effects of androgens are mediated by the Androgen Receptor (AR). Therefore, studies focus on the identification of AR-regulated genes that are also highly expressed in the prostate. STAMP family genes STAMP1/STEAP2 and STAMP2/STEAP4 have only expressed in androgen receptor-positive cells, the role of AR in STAMP family gene expression is an important question. STEAP (Six Transmembrane Epithelial Antigen of Prostate) is the first characterized prostate of enriched six transmembrane gene, expressed in metastatic prostate cancer samples, it is tempting to speculate that STAMP/STEAP family genes may be involved in similar functions with a role for both the normal biology and pathophysiology of the prostate. Using siRNA technology in LNCaP cells expressing STAMP genes per se, an apoptosis panel including pro-apoptotic and/or apoptotic molecules was assayed by RT-PCR, By this research project, prostate-specific STAMP gene family and its regulatory effects on the p53- and caspase-related pathways were characterized.

Grafting of recovered shoots reduces bois noir disease incidence in vineyard

Bois noir (BN) disease of grapevine is associated with ‘Candidatus Phytoplasma solani’ (CaPsol). Due to its multifaceted ecology, BN control is extremely difficult. Several studies showed that BN recovery can be elicited by abiotic stresses and treatment with resistance inducers. In this study, field trials along with molecular analyses have been conducted to evaluate if grafting of recovered shoots can increase the BN recovery rate in symptomatic grapevines and decrease the new CaPsol infection rate on asymptomatic grapevines. Field trials were performed in two vineyards (cv. Chardonnay/Kober 5BB) in Franciacorta (Lombardy Region, northern Italy). Grafting effects were evaluated by symptom observation and CaPsol detection by nested-PCR stamp gene amplification for three consecutive years and compared with non-grafted control vines. Obtained data showed that BN incidence was lower in grafted plants, mainly due to a statistically significant increase of recovery rate, four times higher than in non-grafted plants. These data indicated that grafting of recovered shoots can efficiently induce BN recovery, opening an interesting scenario for its utilization in sustainable strategies of vineyard management.

Multilocus Genotyping of ‘Candidatus Phytoplasma Solani’ Associated with Grapevine Bois Noir in Iran

Simple SummaryUnderstanding the epidemiological cycle of Bois noir (BN) associated with ‘Candidatus Phytoplasma solani’ is vital to managing the disease effectively. The genotyping of ‘Ca. P. solani’ strains, according to tuf, vmp1, and stamp genes, contributes to a better knowledge of the geographical distribution of BN. In this study, we provide information on the molecular variants of ‘Ca. P. solani’ in Iranian vineyards. We observed six known vmp1 variants and discovered two new ones, V24 and V27. This information is useful for future investigations to more accurately understand the epidemiological cycle(s) of BN in Iranian vineyards, contributing to the management of the disease.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.

Geographical and Temporal Diversity of 'Candidatus Phytoplasma solani' in Wine-Growing Regions in Slovenia and Austria.

As the causal agent of the grapevine yellows disease Bois noir, ‘Candidatus Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple “Ca. P. solani” host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf, secY, stamp, and vmp1 genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the tuf gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian–Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and H. obsoletus associated with ‘Ca. P. solani,' which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes.

Responses Taken by Silencing of NFkappaB, STAMP1 and STAMP2 Genes and Expression of NFkB, Act-1, p53 and p73 at -/+ TNFalpha Induced LNCaP Cells

Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer mortality in men in the Western World. The effects of androgens are mediated by the Androgen Receptor (AR). Therefore, studies focus on the identification of AR-regulated genes that are also highly expressed in the prostate. STAMP family genes STAMP1/STEAP2 and STAMP2/STEAP4 have only expressed in androgen receptor-positive cells, the role of AR in STAMP family gene expression is an important question. STEAP (Six Transmembrane Epithelial Antigens of Prostate) is the first characterized prostate enriched six transmembrane genes, expressed in metastatic prostate cancer samples, it is tempting to speculate that STAMP/STEAP family genes may be involved in similar functions with a role for both the normal biology and pathophysiology of the prostate. Using siRNA technology in LNCaP cells expressing STAMP genes per se, an apoptosis panel including pro-apoptotic and/or apoptotic molecules was assayed by RT-PCR. In this research project, the prostate-specific STAMP gene family and its regulatory effects on the nuclear factor kappa B and caspase-related pathways were characterized. Considering that the beta-actin response in the control group was high in the immunolabeling studies, an increase in the induction of Tumor Necrosis Factor (TNF) was detected in the signals received with the vital proteins NFkB and akt, which were silenced by siRNA, which means that STAMP genes potentiate vital proteins.

Presence and seed transmission of phytoplasmas in tomato fields in Italy

Tomatoes are the second most consumed vegetable, quite often severely affected by phytoplasma-associated diseases worldwide. The identification and confirmation that the phytoplasmas associated with the disease in Southern Italy are also transmitted through seeds was achieved. DNA extractions from mother plants collected in 2020 and 2021 and the seedlings from these plants, tested at different times, allowed the detection of phytoplasmas in both mother plants and seedlings grown under an insect-proof greenhouse. Phytoplasmas enclosed in groups 16SrX-B (‘Candidatus Phytoplasma prunorum’) and 16SrV-A (‘Ca. P. ulmi’) were identified in 2020 samples, and the same phytoplasma groups were identified in the seedlings. Phytoplasmas enclosed in groups 16SrI and 16SrXII were identified in mother plants collected in 2021 and were also identified in the seedlings. Moreover, a difference in germination percentage was observed. The 16SrXII-A group phytoplasmas detected in the mother plants and were identified as variants St5 for the stamp gene, tuf-b1 for the tuf gene and Vm44 for the vmp1 gene. This research is a significant confirmation of previous laboratory results on transmission of phytoplasmas through seeds from several diseased plant species. The epidemiological role of phytoplasma seed transmission is an essential knowledge for efficient management strategy planning for a seed production aimed at reducing the impact of phytoplasma-associated diseases in horticultural relevant crops such as tomatoes.

Molecular and spatial analyses reveal new insights on Bois noir epidemiology in Franciacorta vineyards

AbstractBois noir (BN) grapevine disease is associated with ‘Candidatus Phytoplasma solani’ (CaPsol), a pathogen with a complex ecology including multiple insect vectors and plant hosts. A key point to improve the effectiveness of BN control strategies consists in determining the epidemiological role of ground‐cover weeds. The present study employed a multidisciplinary approach, based on the application of spatial (spatial analysis by distance indices) and molecular (stamp gene typing) analyses, to identify weeds with a potential role in BN epidemiology in northern Italy. Generated data showed that in addition to Convolvulus arvensis, one of the main CaPsol inoculum sources, Chenopodium album, Polygonum aviculare and Trifolium repens were found associated with BN epidemiology. CaPsol molecular typing highlighted that the strains prevalent in symptomatic grapevines were characterised by stamp sequence variants St19, St11 (nettle‐related) and St5 (bindweed‐related). The latter was prevalent also in Hyalesthes obsoletus and weeds, suggesting their main association with bindweed‐related epidemiology. On the other hand, nettle‐related CaPsol strains were occasionally found in H. obsoletus and weeds. Considering that H. obsoletus‐mediated transmission of CaPsol occurs mainly with young instars, further investigations will confirm if, in addition to bindweed and nettle, weeds associated with BN epidemiology in Franciacorta can represent larval developmental hosts and, consequently, act as CaPsol reservoirs for transmission to grapevine. Moreover, other studies are needed to clarify the relationship between such weeds and CaPsol alternative vectors.

Symptomatology, (Co)occurrence and Differential Diagnostic PCR Identification of 'Ca. Phytoplasma solani' and 'Ca. Phytoplasma convolvuli' in Field Bindweed.

Field bindweed (Convolvulus arvensis) is one of the major natural plant hosts and reservoirs of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), the causal agent of plant diseases in diverse agricultural crops, including Bois noir (BN) disease of grapevine. Phylogenetically, the most closely related phytoplasma to ‘Ca. P. solani’, the ‘Ca. P. convolvuli’, induces disease in field bindweed that is known by its symptoms as bindweed yellows (BY). The occurrence, coinfection and symptoms association of the two phytoplasmas in shared host plants were the subject of this study. Specific primers for the amplification of the elongation factor Tu gene (tuf) were developed for the identification of ‘Ca. P. convolvuli’ (by conventional nested PCR), as well as primers for simultaneous detection of ‘Ca. P. solani’ and ‘Ca. P. convolvuli’ by duplex SYBR Green real-time PCR. Among symptomatic bindweed plants, 25 and 41% were infected with a single phytoplasma species, ‘Ca. P. solani’ and ‘Ca. P. convolvuli’, respectively, while 34% were infected with both phytoplasmas. None of the non-symptomatic control plants carried phytoplasma, while non-symptomatic plants from our previous epidemiological studies in BN-affected vineyards were confirmed to be infected solely with ‘Ca. P. solani’. Stamp gene typing revealed Rqg50 and Rqg31 ‘Ca. P. solani’ genotypes in plants coinfected with ‘Ca. P. convolvuli’, while three diverse genotypes (Rqg50, GGY and Rpm35) were identified in a single locality with symptomatic bindweeds infected solely with ‘Ca. P. solani’. Variations in symptoms and their association with each of the phytoplasmas are described and documented. The symptom of bushy appearance could be single out as specific for ‘Ca. P. convolvuli’ infection, while occurrence of ‘Ca. P. solani’ could not be unequivocally associated with specific alterations in infected bindweeds. The results are discussed in the context of the epidemiological and ecological complexity of ‘Ca. P. solani’-induced diseases and the relationship between the two phytoplasma relatives in shared host plant.

Rubbery Taproot Disease of Sugar Beet in Serbia Associated with 'Candidatus Phytoplasma solani'.

Rubbery taproot disease (RTD) of sugar beet was observed in Serbia for the first time in the 1960s. The disease was already described in neighboring Bulgaria and Romania at the time but it was associated with abiotic factors. In this study on RTD of sugar beet in its main growing area of Serbia, we provide evidence of the association between 'Candidatus Phytoplasma solani' (stolbur phytoplasma) infection and the occurrence of typical RTD symptomatology. 'Ca. P. solani' was identified by PCR and the sequence analyses of 16S ribosomal RNA, tuf, secY, and stamp genes. In contrast, the causative agent of the syndrome "basses richesses" of sugar beet-namely, 'Ca. Arsenophonus phytopathogenicus'-was not detected. Sequence analysis of the stolbur strain's tuf gene confirmed a previously reported and a new, distinct tuf stolbur genotype (named 'tuf d') that is prevalent in sugar beet. The sequence signatures of the tuf gene as well as the one of stamp both correlate with the epidemiological cycle and reservoir plant host. This study provides knowledge that, for the first time, enables the differentiation of stolbur strains associated with RTD of sugar beet from closely related strains, thereby providing necessary information for further epidemiological work seeking to identify insect vectors and reservoir plant hosts. The results of this study indicate that there are differences in hybrid susceptibility. Clarifying the etiology of RTD as a long-known and economically important disease is certainly the first step toward disease management in Serbia and neighboring countries.

Identification and ecology of alternative insect vectors of \u2018Candidatus Phytoplasma solani\u2019 to grapevine

Bois noir, a disease of the grapevine yellows complex, is associated with ‘Candidatus Phytoplasma solani’ and transmitted to grapevines in open fields by the cixiids Hyalesthes obsoletus and Reptalus panzeri. In vine-growing areas where the population density of these vectors is low within the vineyard, the occurrence of bois noir implies the existence of alternative vectors. The aim of this study was to identify alternative vectors through screening of the Auchenorrhyncha community, phytoplasma typing by stamp gene sequence analyses, and transmission trials. During field activities, conducted in Northern Italy in a vineyard where the bois noir incidence was extremely high, nine potential alternative insect vectors were identified according to high abundance in the vineyard agro-ecosystem, high infection rate, and harbouring phytoplasma strains characterized by stamp gene sequence variants found also in symptomatic grapevines. Transmission trials coupled with molecular analyses showed that at least eight species (Aphrodes makarovi, Dicranotropis hamata, Dictyophara europaea, Euscelis incisus, Euscelidius variegatus, Laodelphax striatella, Philaenus spumarius, and Psammotettix alienus/confinis) are alternative vectors of ‘Candidatus Phytoplasma solani’ to grapevines. These novel findings highlight that bois noir epidemiology in vineyard agro-ecosystems is more complex than previously known, opening up new perspectives in the disease management.

Prevalence of a ‘Candidatus Phytoplasma solani’ strain, so far associated only with other hosts, in Bois Noir‐affected grapevines within Tuscan vineyards

Due to its complex epidemiological cycle, including several polyphagous insect vectors and host plants, and the absence of efficient control strategies, Bois Noir (BN) disease of grapevine is encroaching wider territories in the main viticultural areas worldwide. Molecular approaches allowed to increase the knowledge about its etiological agent (Bois Noir phytoplasma, BNp; ‘Candidatus Phytoplasma solani’ species), revealing interesting features concerning BNp population structure and dynamics and transmission routes in vineyard agro‐ecosystems. In the present study, a multilocus sequence typing approach (vmp1 and stamp genes) was utilised for describing the genetic diversity among BNp strain populations in 17 vineyards localised in two distinct geographic areas in Tuscany (central Italy). The results confirmed that BNp ecology in Tuscan vineyards is mainly associated to the bindweed‐related host system, and allowed the identification of 14 BNp vmp1/stamp genotypes. Interestingly, the prevalent genotype (Vm43/St10) was never found in grapevines outside of Tuscany. Moreover, statistical analyses showed significant differences between the composition of BNp strain populations identified in grapevines from north‐western and central‐eastern Tuscany. These results reinforce the hypothesis that distinct geographic areas, probably associated with different ecological niches, can drive the selection of BNp strains, also favouring the entrance of unusual ‘Ca. Phytoplasma solani’ genotypes in vineyards.

Molecular identification and characterization of phytoplasmas infecting tomato in North Italy

The present work aimed to investigate the aetiology of a disease affecting tomato in Oltrepo pavese (Lombardy, North Italy). During field surveys carried out in a tomato plantation in August 2015, symptoms typically associated with phytoplasma infection (leaf purpling, witches’-broom, flower and fruit alterations, and stunting) were observed on tomato plants. Based on 16S rRNA gene amplification and nucleotide sequence analyses, ‘Candidatus Phytoplasma solani’ was detected in 82% of symptomatic tomato plants and in bindweed plants. Molecular characterization based on tufB and stamp gene nucleotide sequence analyses revealed the presence of two 'Ca. P. solani' strain types: the type tufB-b/St5, identified respectively in 83% and 100% of tomato and bindweed infected plants, and the type tufB-a/St18 identified in 17% of tomato infected plants. These results suggest that ‘Ca. P. solani’ ecology in the examined agro-ecosystem is associated mainly with the bindweed-related host system. Based on such evidences, it should be interesting to evaluate the incidence dynamics of the disease and to investigate its epidemiology by tracing the movements of ‘Ca. P. solani’ throughout neighbouring fields.

Important genetic diversity of ‘Candidatus Phytoplasma solani’ related strains associated with bois noir grapevine yellows and planthoppers in Azerbaijan

Bois noir (BN) is an important grapevine yellows endemic to the Euro-Mediterranean basin caused by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), a non culturable plant pathogenic Mollicute. Bois noir symptoms could be associated with ‘Ca. P. solani’ in two Azerbaijanian vineyards where disease incidence and severity were recorded for five local Vitis vinifera cultivars. In order to gain insight into the epidemiology of Bois noir in Azerbaijan, ‘Ca. P. solani’ isolates infecting plants were characterized by multi-locus sequence analysis and their secY and stamp gene sequences compared to that of the strains detected in other plants and in local Cixiidae planthoppers. Genotypes were determined for two non-ribosomal house-keeping genes, namely tuf and secY, as well as two variable markers namely Stamp and mleP1 genes, that respectively encode the antigenic membrane protein AMP and a 2-Hydroxycarboxylate transporter. The Azerbaijanian BN phytoplasma isolates corresponded to three tufB and secY genotypes. A finer differentiation of Azerbaijanian ‘Ca. P. solani’ isolates was obtained with mleP1 as five different mleP1 genetic variants were found. Finally, Stamp gene allowed differentiating four new genotypes in grapevine among the 10 new Stamp genotypes detected in various plants in Azerbaijan. The preliminary survey for infected insects conducted in northern Azerbaijan, led to the identification of Hyalesthes obsoletus and Reptalus noahi as potential vectors for two ‘Ca. P. solani’ new genotypes phylogenetically distant from the known genetic clusters. Altogether these results indicate an important genetic diversity of BN phytoplasmas in Azerbaijan that certainly result from spread through local insect vectors.

Molecular Typing of Bois Noir Phytoplasma Strains in the Chianti Classico Area (Tuscany, Central Italy) and Their Association with Symptom Severity in Vitis vinifera 'Sangiovese'.

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.

FIRST REPORT OF ‘CANDIDATUS PHYTOPLASMA SOLANI’ IN PEPPER AND CELERY IN BOSNIA AND HERZEGOVINA

In August 2015, in Bijeljina (Semberija province of Bosnia and Herzegovina) pepper and celery plants were surveyed for phytoplasma infections. During the survey pepper plants (Capsicum annuum ) of cvs Fortesa, Niska Sipka and Amanda showing stunting and leaf yellowing were observed, while celery plants (Apium graveolens L.) variety ‘Giant Prague’, expressed leaf whitening and stunting; the percentages of symptomatic plants ranged from 20 to 30%, respectively. Leaf samples from symptomatic and asymptomatic plants were tested to verify phytoplasma presence using nested-PCR/RFLP analyses. Results of 16S rDNA RFLP analyses with Tru1I and tuf typing using HpaII (Langer and Maixner, 2004) indicated the presence of a ‘Candidatus Phytoplasma solani’-related strain in all the symptomatic samples tested. There was no variability in the tuf gene and only tuf-type b was detected. Four symptomatic pepper and two celery samples were selected for further characterization amplifying vmp1 and stamp genes (Fialova et al., 2009; Fabre et al., 2011). The nucleotide sequences of the obtained amplicons were submitted to GenBank (accession Nos. KU340846-51 and KU295501-06 for vmp1 and stamp sequences, respectively). Homology with ‘Ca. P. solani’ sequences from database was 96- 100% for the vmp1 and 99-100% for the stamp gene. Combined RFLP analyses using Tru1I and Hpy188I on stamp and RsaI on vmp1 genes distinguished four ‘Ca. P. solani’ lineages indicating the presence of genetic variability among the phytoplasmas studied. This is the first report of ‘Ca. P. solani’ presence in symptomatic pepper and celery in Bosnia and Herzegovina.

Identification and Characterization of New 'Candidatus Phytoplasma solani' Strains Associated with Bois Noir Disease in Vitis vinifera L. Cultivars Showing a Range of Symptom Severity in Georgia, the Caucasus Region.

Evidence from a preliminary survey highlighted that 'Candidatus Phytoplasma solani', the etiological agent of bois noir (BN) disease of grapevine, infects grapevine varieties in Georgia, a country of the South Caucasus. In this study, field surveys were carried out to investigate the BN symptom severity in international and Georgian native varieties. 'Ca. P. solani' was detected and identified by polymerase chain reaction-based amplification and restriction fragment length polymorphism analysis of 16S ribosomal DNA, and further characterized by multiple gene typing analysis (vmp1 and stamp genes). Obtained data highlighted that the majority of Georgian grapevine varieties showed moderate and mild symptoms, whereas international cultivars exhibited severe symptoms. Molecular characterization of 'Ca. P. solani' from grapevine revealed the presence of 11 distinct phytoplasma types. Only one type (VmGe12/StGe7) was identical to a strain previously reported in periwinkle from Lebanon; the other 'Ca. P. solani' types are described here for the first time. Phylogenetic analyses of vmp1 and stamp gene concatenated nucleotide sequences showed that 'Ca. P. solani' strains in Georgia are associated mainly with the bindweed-related BN host system. Moreover, the fact that 'Ca. P. solani' strains are distributed in grapevine cultivars showing a range of symptom intensity suggests a different susceptibility of such local cultivars to BN.

Vmp1 and stamp genes variability of ‘Candidatus phytoplasma solani’ in Bosnian and Herzegovinian grapevine

Bois noir is one of the most important diseases caused by ‘Candidatus Phytoplasma solani’ affecting grapevine in the Euro-Mediterranean area. ‘Ca. P. solani’ characterization plays a significant role in its diagnosis, control and understanding of its epidemiology. In the present study, two genes encoding membrane proteins, namely vmp1 and stamp, were used to assess the genetic variability of 18 ‘Ca. P. solani’ grapevine isolates from the Herzegovina region (B&H), previously characterised as tuf-b type. Nested-PCR/RFLP and sequence analyses of vmp1 gene showed the presence of three profiles V17, V14 and V4 where V17 was highly prevalent. Phylogenetic analysis of stamp gene revealed the presence of four different genotypes, where Rqg50 was the most frequent. Finally, the combined results of tuf, vmp1 and stamp genes, reported three previously identified genotypes from Serbia, Austria and Montenegro, and two newly described ones (DB1 and DB2). Although performed only on ‘Ca. P. solani’ isolates from infected grapevines, this study provides useful molecular information to get better insight on BN epidemiology in B&H.

Molecular tracing of the transmission routes of bois noir in Mediterranean vineyards of Montenegro and experimental evidence for the epidemiological role of Vitex agnus‐castus (Lamiaceae) and associated Hyalesthes obsoletus (Cixiidae)

Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.

The molecular epidemiology of bois noir grapevine yellows caused by ‘Candidatus Phytoplasma solani’ in the Republic of Macedonia

Bois noir (BN), which is induced by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), is an important grapevine yellows disease that causes severe damage in viticultural regions throughout the Euro-Mediterranean basin. An epidemiological survey to determine potential insect vectors and the primary reservoir plants of BN phytoplasma in Macedonian vineyards was undertaken between 2012 and 2013 in the southeastern part of the country. A study on the species diversity from the suborder Auchenorrhycha revealed the prevalence of the principal vector of ‘Ca. P. solani’, which is the planthopper Hyalesthes obsoletus. Reptalus panzeri, which is the second-most documented BN vector, was not recorded in Macedonian vineyards. Three leafhopper species, namely Psammotettix alienus, Artianus manderstjernii and Euscelis incisus, were also widespread in the BN-affected vineyards, but only H. obsoletus tested positive for ‘Ca. P. solani’. Molecular characterizations were performed by the sequencing and/or RFLP typing of tuf, vmp1 and stamp genes, and the results were used to gain detailed insight into the molecular diversity of the ‘Ca. P. solani’ strains associated with grapevines, tentative reservoir plants (Urtica dioica and Convolvulus arvensis) and the H. obsoletus associated with these plants. Among the 91 ‘Ca. P. solani’ strains detected in diverse plant and insect hosts, three tuf, five vmp1 and 11 distinct stamp genotypes were identified. Twelve comprehensive genotypes of ‘Ca. P. solani’ were detected according to the tuf/vmp1/stamp genotyping. The highest diversity of genotypes was detected among the strains from H. obsoletus individuals associated with U. dioica, of which the most frequent genotype was tuf-ab/V18/M1 (43 %). The tuf-b/V2-TA/STOL comprehensive genotype was found in 33 % of naturally infected grapevines. Two ‘Ca. P. solani’ genotypes were associated with U. dioica, namely (i) tuf-ab/V18/M1 (60 %) and tuf-a/V3/M4 (40 %), and only one genotype (tuf-b/V2-TA/Rqg50) was associated with C. arvensis.