Bois Noir Phytoplasma Research Articles

Versatile physiological functions of the Nudix hydrolase family in berry development and stress response in grapevine

Nudix hydrolases are widely distributed across all classes of organisms and provide the potential capacity to hydrolyze a wide range of organic pyrophosphates. Although Nudix hydrolases are involved in plant detoxification processes in response to abiotic and biotic stresses, the biological functions of Nudix hydrolases remain largely unclear in grapevine. In the present study, a total of 25 putative grapevine Nudix hydrolases (VvNUDXs) were identified by bioinformatics analysis and classified into eight subfamilies based to their preferred substrates. Both tandem and segmental duplications were responsible for the evolution and expansion of the NUDX gene family in grapevine. To investigate the regulatory roles of VvNUDX genes during growth and development, as well as in response to abiotic and biotic stresses in grapevine, the expression patterns were revealed in publicly available microarray data. The spatial and temporal expression patterns of the VvNUDX genes indicated that they might play important roles in multiple developmental processes. Transcriptome and qRT-PCR analyses showed that ten VvNUDX genes were specifically expressed in grapevine berries, suggesting potential roles in grapevine berry development. Expression and phylogenetic analyses demonstrated that VvNUDX1 and VvNUDX3 might be involved in terpenoid biosynthesis in grapevine. Furthermore, most VvNUDX genes active toward the ADP-ribose/NADH showed different patterns in response to various abiotic and biotic stresses, such as salinity and drought, as well as different types of biotic treatments, such as Erysiphe necator, Bois Noir phytoplasma and leaf-roll-associated virus-3 (GLRaV-3). These results indicated that VvNUDX genes were associated with plant detoxification processes in response to abiotic and biotic stresses, and regulate the disease immunity and resistance pathways. The information obtained here may provide good opportunities to explore the physiological functions of VvNUDX genes in berry development and stress response networks in grapevine.

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.

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.

Jasmonate-mediated defence responses, unlike salicylate-mediated responses, are involved in the recovery of grapevine from bois noir disease

BackgroundBois noir is an important disease of grapevine (Vitis vinifera L.), caused by phytoplasmas. An interesting, yet elusive aspect of the bois noir disease is “recovery”, i.e., the spontaneous and unpredictable remission of symptoms and damage. Because conventional pest management is ineffective against bois noir, deciphering the molecular bases of recovery is beneficial. The present study aimed to understand whether salicylate- and jasmonate-defence pathways might have a role in the recovery from the bois noir disease of grapevine.ResultsLeaves from healthy, bois noir-diseased and bois noir-recovered plants were compared, both in the presence (late summer) and absence (late spring) of bois noir symptoms on the diseased plants. Analyses of salicylate and jasmonate contents, as well as the expression of genes involved in their biosynthesis, signalling and action, were evaluated. In symptomatic diseased plants (late summer), unlike symptomless plants (late spring), salicylate biosynthesis was increased and salicylate-responsive genes were activated. In contrast, jasmonate biosynthesis and signalling genes were up-regulated both in recovered and diseased plants at all sampling dates. The activation of salicylate signalling in symptomatic plants might have antagonised the jasmonate-mediated defence response by suppressing the expression of jasmonate-responsive genes.ConclusionsOur results suggest that grapevine reacts to phytoplasma infection through salicylate-mediated signalling, although the resultant full activation of a salicylate-mediated response is apparently ineffective in conferring resistance against bois noir disease. Activation of the salicylate signalling pathway that is associated with the presence of bois noir phytoplasma seems to antagonise the jasmonate defence response, by failing to activate or suppressing both the expression of some jasmonate responsive genes that act downstream of the jasmonate biosynthetic pathway, as well as the first events of the jasmonate signalling pathway. On the other hand, activation of the entire jasmonate signalling pathway in recovered plants suggests the potential importance of jasmonate-regulated defences in preventing bois noir phytoplasma infections and the subsequent development of bois noir disease. Thus, on one hand, recovery could be achieved and maintained over time by preventing the activation of defence genes associated with salicylate signalling, and on the other hand, by activating jasmonate signalling and other defence responses.

Test performance study of isothermal amplification tests for the detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’

This test performance study (TPS) was carried out on DNA samples from grapevine, clematis, fungi and bacteria to compare and validate loop‐mediated isothermal amplification (LAMP) tests for detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (Grapevine Bois noir phytoplasma). Two LAMP tests, for Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (as developed by Kogovšek and colleagues), with proven applicability for rapid laboratory or on‐site detection were included in this study. They were performed in 10 laboratories. In addition, the commercial Qualiplante/Hyris isothermal amplification test for Grapevine flavescence dorée phytoplasma was performed in three laboratories. The accuracy of the three tests was shown to be over 98%. Moreover, the high accuracy of these tests, which used different devices across different laboratories, confirmed their reproducibility.

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for ‘Candidatus Phytoplasama solani’ (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterranean Plant Protection Organisation recommendations. In addition, 286 grapevine leaf samples from the 2015 growing season were tested with this new real-time LAMP assay and an assay previously developed for detection of Flavescence dorée phytoplasma (FDp). These LAMP assays for detection of both BNp and FDp used without any DNA extraction step, which is a required step for qPCR analysis, were comparably effective to qPCR, and positive results were obtained in less than 35 min.

A regional-scale survey to define the known and potential vectors of grapevine yellow phytoplasmas in vineyards South of Swiss Alps

The most important Grapevine Yellows (GY) phytoplasma diseases in Europe are Flavescence doree (FD) and Bois noir (BN); they are spread in vineyard by two proven vectors, Scaphoideus titanus Ball and Hyalesthes obsoletus Signoret, respectively. Other potential vectors of GY have been identified, which are thought to play a secondary role. The GY control strategies are not always effective and an in-depth study on the ecological cycle of the pathogens at regional scale would be of paramount importance. This study was carried out in 48 representative sites of wine-growing area South of Swiss Alps, with the aim to identify known and potential vectors and to characterize the FD and BN phytoplasmas isolates. Out of 167 Auchenorrhyncha species recorded, 27 were known or potential vectors of phytoplasmas and five of those tested positive for phytoplasmas. S. titanus was infected by 16SrV-D subgroup phytoplasma and no clear relationship between its population density and disease outbreaks was observed. Orientus ishidae harboured 16SrV-C and 16SrV-D subgroups suggesting its potential role in spreading 16SrV-C phytoplasma isolates from arboreal plants to grapevine and FD-D from grapevine to grapevine. H. obsoletus was infected by BN phytoplasmas, tuf-types a and b, however it was collected with relatively low abundance. Reptalus panzeri and R. cuspidatus tested positive to tuf-type b, but only R. cuspidatus was common and abundant in the investigated vineyards. To define the range of alternative vectors using a detailed approach on regional scale provides background information to get a more clear vision on the spreading of phytoplasmas in the vineyards.

Temporal shifts of Bois Noir phytoplasma types infecting grapevine in South Tyrol (Northern Italy)

Results and Discussion: Two different restriction enzyme profiles of the Bois noir (BN) phytoplasma, corresponding to VK-Type I and VK-Type II (LANGER and MAIXNER 2004), were found in South Tyrol (Table). Both these BN-types were previously detected in German viticultural areas affected by grapevine yellows. While in Germany also a third BN-type was described (VK-Type III), which is exclusively restricted to the Mosel valley, this strain was not found in South Tyrol. Each BN-type has been associated with a different herbaceous host plant species of the vectoring insect H. obsoletus: VK-Type I with Urtica dioica, VK-Type II with Convolvulus arvensis, and VK-Type III with Calystegia sepium (LANGER and MAIXNER 2004). Consequently, it

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.

A case study of FD and BN phytoplasma variability in Croatia: multigene sequence analysis approach

Uncultivable bacteria from the genus ‘Candidatus Phytoplasma’ are associated with grapevine yellows (GY) diseases worldwide. In Euro-Mediterranean viticultural areas, GY are most frequently caused by Bois Noir (BN) and Flavescence Doree (FD) phytoplasmas. Surveys of GY in Croatia have been conducted regularly since 1997. BN phytoplasmas have been found to be widespread, while FD phytoplasmas were recently discovered in restricted areas of the country. The aim of this study was to assess the variability of genotypes involved in GY pathosystems by a multilocus sequence typing (MLST) approach. Grapevine, weed and insect vector samples were collected from three locations. Species-specific stamp and vmp1 genes, together with house-keeping genes tuf and secY, were amplified from BN strains. In FD strains, the genes secY, map and uvrB-degV were analyzed. MLST revealed a diversity of BN genotypes, one of which was prevalent and identified in samples of grapevine and the insect vector Hyalesthes obsoletus, corroborating their affiliation to the same pathosystem. Distinct BN strains found in bindweed and two grapevine samples indicated the presence of different BN pathosystems involving a yet unidentified vector, possibly from the genus Reptalus. Moreover, a co-occurence of BN and FD phytoplasma in the same vineyard was identified. The genotyping of FD strains from both grapevine and Scaphoideus titanus showed the presence of at least two distinct FD genotypes at two different locations, suggesting separate introductions of the disease into the country. In this study, MLST proved to be a useful and informative tool in advancing the understanding of GY epidemiology in Croatia.

Bois noir phytoplasma variability in a Mediterranean vineyard system: new plant host and putative vectors

The presence of Bois noir phytoplasma (BNp, Stolbur, 16SrXII-A) in Sicily, one of the most important Italian grape-producing regions, has been known since 1996, but its epidemiology and distribution in the region have been studied only recently. Grapevines and weeds showing symptoms of phytoplasma infection were collected in an organic Chardonnay vineyard, chosen as model vineyard, and tested to verify BNp presence. Leaf- and plant-hoppers were also captured in the same field and included in the analysis. Plant and insect infections were assessed by nested PCR with universal and group-specific (16SrI and –V) phytoplasma primer pairs. BNp was detected in the weed species Erigeron bonariensis, which was then determined as a new natural host of this phytoplasma, and in several Auchenorrhyncha species besides Hyalesthes obsoletus, which can be regarded as new potential vectors. To further evaluate the genetic variability of BNp among host plants and potential vectors, PCR-RFLP of the Stolbur phytoplasma-specific vmp1 gene was carried out. The V1 profile was the most representative, other profiles (V4, V9, V10 and V11) were also detected.

Investigation on ‘bois noir’ epidemiology in north‐eastern Italian vineyards through a multidisciplinary approach

AbstractA multidisciplinary approach, based on field surveys, molecular biology techniques, and spatial data analyses, was utilised to investigate the Bois noir (BN) epidemiology in north‐eastern Italian vineyards during the years 2010–12. Symptomatic grapevines, weeds and specimens of the insect vector Hyalesthes obsoletus were monitored and mapped. Leaf samples from symptomatic grapevines and weeds, and captured insect specimens were analyzed by real‐time PCR to identify BN phytoplasma (BNp; ‘Candidatus Phytoplasma solani’ species), the etiological agent of BN. Data spatial distribution was analyzed using SADIE (Spatial Analysis by Distance IndicEs). Bois noir phytoplasma strains identified in weed candidates for an epidemiological role were characterised by RFLP‐based analyses of tuf gene amplicons. Results highlighted that, in the examined areas, the host systems Convolvulus arvensis – H. obsoletus and Urtica dioica – H. obsoletus play the main role in BN diffusion. It was also evidenced that other weeds (i.e. Chenopodium album and Malva sylvestris) spatially associated with symptomatic grapevines and/or insect vectors and infected by the same tuf type identified in grapevines and insects, could play a role in BN diffusion. On the other hand, some weeds (i.e. Trifolium repens) were uninfected and not associated with symptomatic grapevines and/or insect vectors. The synergic application of our multidisciplinary approach improved the knowledge of BN epidemiology, and provided helpful indication for designing experimental plans to contain BN spreading in vineyards through weed management. The approach described in the present work could be used to investigate the complex epidemiology of other phytoplasma diseases.

First Report of 'Candidatus Phytoplasma solani' and 'Ca. P. convolvuli' Associated with Grapevine Bois Noir and Bindweed Yellows, Respectively, in Georgia.

A survey carried out in Georgian vineyards, located in the Khaketi region, in September 2013, showed the presence of vines of the cultivar Chardonnay with typical grapevine yellows (GY) symptoms including leaf discoloration and curling, berry shriveling, and irregular maturation of wood. In the same vineyards, bindweed (Convolvulus arvensis L.) plants showing shoot proliferation and leaf yellowing were found, suggesting the involvement of phytoplasmas in the disease etiology. Total DNA was extracted by a CTAB method from leaf veins of 18 symptomatic and two asymptomatic grapevines, and from four symptomatic and two asymptomatic bindweeds, and analyzed by PCR assays. Moreover, DNA extracted from 'Candidatus Phytoplasma asteris' strain SAY (group 16SrI), 'Ca. P. solani' strain STOL (group 16SrXII), and 'Ca. P. ulmi' strain EY1 (group 16SrV) were used as positive controls. DNA extracted from healthy periwinkle and a reaction mixture without template were employed as negative controls. Nested PCRs targeting the 16S rDNA, carried out using the primer pairs P1/P7 followed by R16F2n/R16R2 (1), produced a band of the expected size (1,250 nt) in all the symptomatic grapevine and bindweed plants, and in the positive controls. No amplification was observed with DNA from asymptomatic plants nor the negative controls. PCR products were sequenced by a commercial sequencing service (Primm, Milan, Italy). The 16S rDNA nucleotide sequences of phytoplasmas identified in all grapevines and in two bindweed samples shared >99.5% sequence identity with 'Ca. P. solani' reference strain STOL (GenBank Accession No. AF248959), and carried identical STOL-unique signature sequence and distinguishing sequence blocks (3). Moreover, nucleotide sequences of phytoplasmas identified in the other two bindweed samples shared >99.6% sequence identity with 'Ca. P. convolvuli' reference strain BY-S57/11 (JN833705) (2). RFLP and phylogenetic analyses confirmed the affiliation of the phytoplasma strains identified in grapevine and bindweed plants in Georgia to the species 'Ca. P. solani' (subgroup 16SrXII-A) and 'Ca. P. convolvuli' (subgroup 16SrXII-H). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank website with accession nos. KF996535 and KF996536 ('Ca. P. solani' from grapevine and bindweed, respectively), and KF996537 ('Ca. P. convolvuli'). Future studies will focus on investigating the spread and impact of 'Ca. P. solani'-associated bois noir (BN) in Georgia. In particular, the identification of 'Ca. P. solani' in bindweeds suggested the presence of the insect Hyalesthes obsoletus, a polyphagous cixiidae responsible for BN phytoplasma transmission in vineyards in Europe. Accurate surveys and molecular analyses will be performed for identifying the insect vector(s) of the BN associated phytoplasma strains in Georgia. Additional studies will be performed to study the spread and impact of 'Ca. P. convolvuli,' identified only in Italy, Germany, Serbia, and Bosnia and Herzegovina (2), throughout the Caucasian countries.

Incidence of Bois Noir phytoplasma in different viticulture regions of Spain and Stolbur isolates distribution in plants and vectors

The incidence of Bois Noir (BN) disease in grapevine plots, the population of the vector Hyalesthes obsoletus, and the distribution of stolbur phytoplasma isolates were studied over a 4 year period in five regions of Spain. BN incidence in affected plots ranged from 1 to 75 %. A study of the H. obsoletus population indicated that individuals of this insect vector were identified in most of the sampled plots with low populations, from 0.25–5 individuals per aspiration. The population peaks of H. obsoletus were reached at different dates between June 6th and July 24th, depending on the sampled zone and the year. The percentage of H. obsoletus individuals carrying the phytoplasma showed an average of 55 %. In Aragon, this percentage rose to 76 %. The strain tuf-b of stolbur phytoplasma was the most prevalent type in grapevine plants and H. obsoletus, except in grapevine plants from La Rioja where the strain tuf-a was detected in most plants. A study of the vmp1 gene revealed the presence of four different isolates in grapevine plants and H. obsoletus.

First Report of 'Candidatus Phytoplasma solani' Strains Associated with Grapevine Bois Noir in Jordan.

During a survey carried out in Jordanian vineyards in August and October 2012, grapevine (Vitis vinifera L.) plants showing typical grapevine yellows (GY) disease symptoms, including leaf discoloration and curling, berry shriveling, and irregular maturation of wood, were observed. In the same vineyards, bindweed (Convolvulus arvensis L.) plants showing stunting and leaf chromatic alteration were found, suggesting the involvement of phytoplasmas in the disease etiology. Using a CTAB method, total DNA was extracted from leaf veins of 25 symptomatic and two asymptomatic grapevines, and from five symptomatic and two asymptomatic bindweeds for PCR analysis. DNAs from periwinkle (Catharanthus roseus (L.) G. Don) plants infected by 'Ca. Phytoplasma asteris' strain SAY (group 16SrI), 'Ca. Phytoplasma solani' strain STOL (group 16SrXII), and 'Ca. Phytoplasma ulmi' strain EY1 (group 16SrV), were used as positive controls. DNAs from healthy periwinkle and reactions without template DNA were employed as negative controls. 16S rDNA nested PCRs, carried out using the primer pairs P1/P7, followed by R16F2n/R16R2 (1), yielded an amplicon of the expected size (1,250-bp) in three grapevine and in five bindweed samples, and in positive controls. Amplicons were not produced with DNA from 22 symptomatic grapevines (probably because samples were collected late in the growing season and phytoplasma distribution in plants was non-uniform [2]); nor from asymptomatic plants and negative controls. PCR products were sequenced by commercial services in Italy (Primm, Milan) and Korea (Macrogen Inc., Soul). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank with accessions KC835139 (from grapevine) and KC835140 (from bindweed). The 16S rDNA nucleotide sequences of phytoplasmas identified in grapevine and bindweed in Jordan shared >99.5% sequence identity with 'Ca. Phytoplasma solani' reference strain STOL (AF248959), and carried identical STOL-unique signature sequences and distinguishing sequence blocks (3). Phylogenetic and in silico RFLP analyses confirmed the affiliation of phytoplasma strains identified in grapevine and bindweed in Jordan to the species 'Ca. Phytoplasma solani' (subgroup 16SrXII-A), opening an avenue to future studies on the dissemination and impact of Bois noir (BN) in Jordan. These studies may add new information about BN, previously reported in neighboring countries (4). Further studies will investigate the role of Hyalesthes obsoletus Signoret, a polyphagous Cixiidae responsible for the BN phytoplasma transmission in Europe, and other possible insect vector(s) in the BN spread in Jordan.

Elimination of Grapevine Bois Noir Phytoplasma by Tissue Culture Coupled or not With Heat Therapy or Hot Water Treatment

Background and Aims: Bois Noir phytoplasma is one of the most widespread yellows in grape industry in the European and Middle Eastern countries causing damages of increasing concern for the growers. So far, traditional before hot water therapy was not totally successfully in controlling this disease as it interfered with the vitality of woody propagation material leading to a weak recovery of grafted vines. This study examined the efficiency of tissue culture techniques to eliminate Bois Noir phytoplasma from grapevines. Methods: Six tissue culture protocols were tested on Chardonnay grapevine infected by Bois Noir by using stem cuttings and shoot tips associated or not to heat treatment at 38 ± 1°C for 40 days from culture initiation; and stem cuttings combined with a hot water bath at 50°C for 15 and 30 min prior to culture initiation. Key results: The protocols were all suitable, either for shoot regeneration or for phytoplasma elimination. Stem cutting culture coupled with heat or hot water treatments appeared to be the most effective treatments leading to a correct rate of survival explants and yielding to 100% sanitized shoots. Conclusion: On the basis of these results, the stem cutting culture coupled with heat or hot water treatment and followed by micropropagation of sanitized explants, appears to be a good candidate to become a routine technique for producing phytoplasma-free vines within certification programs.

Recovery from stolbur disease in grapevine involves changes in sugar transport and metabolism.

Grapevine can be severely affected by phytoplasmas, which are phytopathogenic Mollicutes invading the sieve elements of the host plant. The biochemical and molecular relationships between phytoplasmas and their hosts remain largely unexplored. Equally unknown is an interesting aspect of the pathogen–plant interaction called “recovery,” which is a spontaneous remission of symptoms in previously symptomatic plants. Recovered plants develop resistance mechanisms correlated with ultrastructural and biochemical changes in the sieve elements. Callose as well as sugars are involved in several plant defense processes and signaling. In the present work we have examined the possible involvement of callose, as well as callose synthase, sugar transporter, and cell wall invertase genes, during the infection and after “recovery” of grapevine from bois noir (BN). Ultrastructural investigation of leaf tissue showed that callose accumulated in the sieve elements of diseased grapevine; moreover, two genes encoding for callose synthase were up-regulated in the infected leaves. Regarding sucrose, expression analysis showed that sucrose transport and cleavage were severely affected by BN phytoplasma, which induced the establishment of a carbohydrate sink in the source leaf, and was analogous to other obligate biotrophs that acquire most of their nutrients from the host plant. Interestingly, whereas in recovered plants the transcript level of sucrose synthase was similar to healthy plants, sucrose transporters as well as cell wall invertase were expressed to a greater degree in recovered leaves than in healthy ones. Recovered plants seem to acquire structural and molecular changes leading to increases in sucrose transport ability and defense signaling.

Quantitative PCR assay for detection of bois noir phytoplasmas in grape and insect tissue

In Europe's vineyards Bois noir (BN) is an expanding yellows disease on Vitis vinifera . It is associated with phytoplasmas of the stolbur group (16SrXII-A). Two subtypes are important, one is associated with Urtica dioica and one with Convolvulus arvensis. Both phytoplasma types are transmitted by the insect Hyalesthes obsoletus . A nucleic acid extraction method for V. vinifera and H. obsoletus was developed together with a real time PCR (qPCR) assay based on a polymorphic sequence with homology to a putative dimethyladenosine transferase. The comparison of the conventional detection method with the qPCR assay of 40 insect and 40 V. vinifera samples showed a 10 % higher sensitivity of qPCR in plant samples. The titer of phytoplasmas in H. obsoletus was 2643-fold increased in the strongest infected samples compared to the lowest ones. The results suggest this real-time PCR as a valid and fast alternative procedure for the detection and quantification of BN phytoplasmas. The assay allows to discriminate the two phytoplasma types and to quantify phytoplasmas in H. obsoletus .

First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia.

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable.