Grapevine Yellows Phytoplasmas Research Articles

Grapevine yellows in Jordan: Associated phytoplasmas, putative insect vectors and reservoir plants

AbstractField surveys were conducted in wine and table grape vineyards from June to October 2020 in 13 locations belonging to five governorates in North and South Jordan. Typical grapevine yellows symptoms, including leaf reddening/yellowing and rolling were observed on 10% to 55% of vines. Nested PCR‐based amplification of the 16S rRNA gene detected phytoplasmas in 22% and 15.7% of the analysed symptomatic wine and table grape cultivar plants, respectively. Amplicon nucleotide sequence analyses identified the detected phytoplasmas as “Candidatus Phytoplasma solani” (taxonomic subgroup 16SrXII‐A), “Ca. P. omanense” (16SrXXIX‐A and ‐B), “Ca. P. aurantifolia” (16SrII‐C) and “Ca. P. asteris” (16SrI‐R) in 72.4%, 17.2%, 6.9% and 3.4% of infected plants, respectively. Such phytoplasmas were found differentially distributed in wine and table grape cultivar vineyards surveyed. Further investigation identified “Ca. P. solani” in the putative insect vectors Orosius cellulosus (first report in Jordan), Euscelidius mundus, Laodelphax striatellus, and Circulifer sp., and in bindweed; “Ca. P. aurantifolia” in the insect O. cellulosus and in bindweed; “Ca. P. omanense” in the insect Psammotettix striatus; and “Ca. P. asteris” in the insects Arboridia adanae, Cicadulina bipunctata, Circulifer sp., L. striatellus, Hyalesthes obsoletus, and P. striatus. Based on this preliminary data, ecological cycles of such phytoplasmas are discussed. Results suggest that the diversity and ecology of grapevine yellows phytoplasmas in Jordan are more complex than previously known, leading to a potential risk of disease outbreaks.

List of non-EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L.

Following a request from the European Commission, the EFSA Panel on Plant Health prepared a list of non‐EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L. A systematic literature review and search of databases identified 27 phytoplasmas infecting one or more of the host genera under consideration. These phytoplasmas were assigned to three categories. The first group (a) consists of 10 non‐EU phytoplasmas, known to occur only outside the EU (‘Candidatus Phytoplasma australiense’, ‘Ca. P. hispanicum’, ‘Ca. P. pruni’‐related strain (NAGYIII), ‘Ca. P. pyri’‐related strain (PYLR) and Buckland valley grapevine yellows phytoplasma) or having only limited presence in the EU (‘Ca. P. aurantifolia’‐related strains, ‘Ca. P. fraxini’, ‘Ca. P. phoenicium’, ‘Ca. P. trifolii’ and ‘Ca. P. ziziphi’). The second group (b) consists of three non‐EU phytoplasmas, whose presence in the target plant species is not fully supported by the available literature. The third group (c) consists of 14 phytoplasmas with substantial presence in the EU (i.e. they are originally described or reported from the EU or known to occur or be widespread in some EU Member States or frequently reported in the EU). Phytoplasmas of categories (b) and (c) were excluded at this stage from further categorisation efforts. One phytoplasma from category (a) (‘Ca. P. phoenicium’) was excluded from further categorisation, as a pest risk assessment has been performed by EPPO. Comments provided by the EU Member States were integrated in the opinion. The main uncertainties of this listing concern: the geographic distribution and prevalence, the taxonomy, biology and host range. The phytoplasmas considered as non‐EU and whose presence in target plant species is fully supported by literature (category (a)) are categorised by the Panel in a separate opinion.

Phytoplasmas associated with grapevine yellows disease in Iran: first report of a ‘Candidatus Phytoplasma trifolii’‐related strain and further finding of a ‘Ca. P. solani’‐related strain

Over recent years, grapevines (Vitis vinifera) with symptoms similar to grapevine yellows disease (severe yellows and redness, inward curling of leaves and stunting) have been observed in vineyards from different regions of Iran, especially Zanjan province (Figs 1-2). Therefore, a survey was conducted in 2018 to identify possible phytoplasma agents. A total of 19 samples from symptomatic (16) and asymptomatic (3) plants (cvs. Keshmeshi Qermez and Askari) were collected from vineyards in the Darsajin, Khoramdareh and Zanjan areas in Zanjan province. Total DNA was extracted (Maixner et al., 4) from the main veins and petioles and nested-PCR with universal primers P1/P7 (Deng & Hiruki, 1) in the first round followed by R16F2n/R2 (Gundersen & Lee, 2) in the nested round was conducted. Nested PCR products were obtained from all symptomatic plants but not from asymptomatic plants. Direct sequencing of the partial 16S rRNA gene from six randomly selected plants confirmed that phytoplasmas associated with grapevine yellows were related to two different ribosomal groups. In BLAST analysis, the sequences obtained (1220 bp) from samples 1, 3 and 5 (GenBank Accession Nos MK392483-MK392485) had 99-100% identity with strains affiliated with ‘Ca. P. trifolii’ (MK15809, MK379605 and MK372596) and samples 6, 7 and 8 (MK392486-MK392488) had 99-100% identity with ‘Ca. P. solani’ related strains (KF583785, MN398472 and MN398467). There was no sequence variation between the three isolates of the 'Ca. P. trifolii'-related strain, or the isolates of the 'Ca. P. solani'-related strain. The percentage sequence identity for the isolates in this study to the reference strain for each species was 99.83% for 'Ca. P. trifolii' (AY390261) and 99.59% for ‘Ca. P. solani’ (AF248959). Phylogenetic analysis was conducted with the maximum likelihood method using MEGA7 software (Kumar et al., 3). According to the phylogenetic tree based upon the 16S rDNA sequences, grapevine yellows phytoplasma strains were clustered in ‘Ca. P. solani’ (16SrXII-A) and ‘Ca. P. trifolii’ (16SrVI-A) (Fig. 3). ‘Ca. P. solani’ and ‘Ca. P trifolii’ -related strains have been reported from many plant species in different areas worldwide. Previous studies reported several ‘Ca. Phytoplasma’ species associated with grapevine yellows disease from Iran including ‘Ca. P. solani’, ‘Ca. P. aurantifolia’, ‘Ca. P. fraxini’, and ‘Ca. P. phoenicium’ (Mirchenari et al., 5; Zamharir et al., 6). To our knowledge, this is the first report of a ‘Ca. P. trifolii‘-related strain associated with grapevine yellows disease in Iran.

First report of grapevine yellows phytoplasma in viticultural areas of the South Brazil

The highlands of Southern Brazil are the most important grapes and wine production region of the country. Diseases are of special concern in viticulture as they are responsible for important losses in yield and quality. Samples from grapevine plants showing yellows symptoms in vineyards of this region were collected and analyzed by nested-PCR. Phytoplasma detected were then classified by in silico PCR/RFLP and phylogenetic analysis. Six out of fifteen symptomatic plants were positive for phytoplasma identified as belonging to the 16SrIII-J subgroup. This is the first report of grapevine yellows association with phytoplasma presence in the of South Brazil.

Occurrence of Scaphoideus titanus Ball and some other Auchenorrhyncha in the vineyards of western Slovakia

A study of Auchenorrhyncha was carried out in 2014 and 2015 in 7 vineyard plots with different varieties and pest management strategies in the Nitra wine region and Lesser Carpathian wine region in western Slovakia. The aim of this study was to obtain information related to the presence of potential vector insects associated with grapevine yellows phytoplasmas from the Flavescence dorée and Bois noir groups. Insects were collected by sweeping with an entomological net. Thirty species of Auchenorrhyncha were identified as belonging to 6 families. Cicadellidae were the most abundant, comprising 20 species. Scaphoideus titanus was collected at 4 localities. Identification of the phytoplasma vector is critical to the national strategy for assessment and control of vectors spreading the phytoplasma disease in Slovakian vineyards. The first finding of Metcalfa pruinosa was noticed in vineyards in Slovakia.

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.

Grapevine yellows: Comparison of different procedures for DNA extraction and amplification with PCR for routine diagnosis of phytoplasmas in grapevine

In order to devise a procedure to be used as reference for detection of grapevine phytoplasmas and monitoring of Flavescence doree, 12 combinations comprising three methods of plant DNA extraction and 4 procedures for amplification in polymerase chain reaction of phytoplasma DNA were examined in parallel using the same plant tissues infected with phytoplasmas. In a first series tissues of periwinkles ( Catharanthus roseus ) infected with phytoplasma isolates of the Elm yellows group (16SrV) and maintained in the greenhouse, were used. In a second series tissues of grapevines ( Vitis vinifera ) naturally infected with Flavescence doree or Palatinate grapevine yellows phytoplasma were used. The DNA preparations obtained with each of the three extraction procedures were used undiluted or serially diluted, as target DNA in the 4 nested-polymerase chain reactions. The results showed differences in the efficiency among different methods of extraction as well as in the sensitivity among the DNA amplification procedures, which improved when DNA extracted from field grapevines was diluted. After additional comparative validation on numerous field-collected samples of GY-affected grapevines, the quickest extraction procedure was selected for use in routine diagnosis, with nested-PCR amplification either of ribosomal DNA or of the FD9 DNA fragment specific for Flavescence doree and other 16SrV group phytoplasmas.

Occurrence and identification of grapevine phytoplasmas in main viticultural regions of Turkey

Intensive surveys were conducted in the main viticultural areas of Turkey in 2009-2010. Two hundred eighty nine symptomatic and 20 non-symptomatic plant samples were collected and subjected to nucleic acid extraction followed by PCR, nested PCR and RFLP analyses to detect phytoplasma presence and for their identification. The incidence rate of phytoplasma infection was 18.33% and the majority of the symptomatic grapevines were infected with grapevine yellows phytoplasmas belonging to 16SrXII-A subgroup (“bois noir”). Phytoplasmas of 16SrV group, aster yellows (16SrI-B) and pigeon pea witches’ broom (16SrIX) groups were also detected in the surveyed vineyards. Phytoplasma-associated infections were present more on wine grapevine cultivars (73.6%), such as Alphonse Lavallee, Alicante Bouschet, Chardonnay, Shiraz, Cabernet Sauvignon, Merlot, Sauvignon Blanc and Pinot Noir, compared to table grapes (26.4%), such as Bogazkere, Sirfani, Tahannebi and Emir.

Descriptive assessment of uncertainties of qualitative real‐time PCR for detection of plant pathogens and quality performance monitoring

In most cases, plant pest diagnostic methods are interpreted as providing qualitative data. However, although qualitative, it is still important to identify all the sources and components of uncertainty in the diagnostic process and to ensure that appropriate measures are taken to reduce the uncertainty and that the form of reporting the result does not give the wrong impression of the uncertainty. This process was illustrated using real‐time PCR testing of grapevine yellows phytoplasma. Some additional quality performance monitoring of real‐time PCR is also described. This approach has been used by the National Institute of Biology, Slovenia, since 2012, in support of accreditation under ISO 17025, and it can also be applied to other detection methods and other pests.

Presence of putative insect vectors of grapevine yellows phytoplasmas in Turkey

Turkey is one of the nations native to grapevine. Main grapevine yellows in Euro-Mediterranean regions are “flavescence doree” and “bois noir”. Phytoplasma associated diseases are phloem limited, transmitted mainly by vectors and grafting and therefore intensive surveys were conducted in major viticultural areas in Marmara, Aegean, Central Anatolia, Eastern, and South Eastern Anatolia regions of Turkey in 2008–2011 in order to determine and to verify the presence of putative grapevine yellows phytoplasma insect vectors. Insects were collected from vineyards and neighbouring weeds and 28 species belonging to eight families of Hemiptera order were identified. Vectors of “bois noir” phytoplasma Euscelis lineolatus Brulle, Hyalesthes obsoletus Signoret and Dictyophara europaea L. were collected whereas Scaphoides titanus Ball., vector of “flavescence doree” phytoplasma was not detected. Results of PCR and nested-PCR assays were negative for phytoplasma presence in all sampled insects.

Flavescence dorée phytoplasma titre in field‐infected Barbera and Nebbiolo grapevines

Flavescence dorée phytoplasma (FDP) titre in two red grapevine cultivars, Barbera and Nebbiolo, was measured over the vegetative seasons of two consecutive years in two vineyards of the Piemonte Region (northwestern Italy), with a double absolute quantification of FDP cells and grapevine DNA in real‐time PCR. The relationships of pathogen concentration to cultivar susceptibility and symptom severity were investigated. FDP titre was always higher in cv. Barbera than in cv. Nebbiolo infected vines, and this difference was significant at early and late summer samplings of 2008 and at early summer sampling of 2009. A seasonal trend in FDP concentration (low in spring, high in early summer and intermediate in late summer) was conserved for cvs Barbera and Nebbiolo in both years and vineyards. Considering both cultivars and years from both vineyards, a significant positive correlation between FDP concentration and symptom severity was found in the spring samples. Regarding the FDP strains (‐C or ‐D), no differences in pathogen titres were detected for either cultivar. Similarly, the presence of another grapevine yellows phytoplasma, bois noir, a subgroup 16SrXII‐A phytoplasma, in mixed infection with FDP strains had no effect on FDP concentration. These results demonstrate for the first time that grapevine cultivars with different susceptibility to FDP support different pathogen titres.

Screening for phytoplasma presence in West Herzegovina vineyards

Surveys for grapevine yellows phytoplasmas in Bosnia and Herzegovina have been conducted since 2004 and vineyards in Srpska region where mostly monitored. In 2011 an extended survey was done in vineyards of West Herzegovina, the most important region for grapevine industry. Four areas were inspected for presence of phytoplasma symptoms and 57 grapevine and weed samples were collected for laboratory analyses. Nested-PCR/RFLP analyses allow to verify presence of ‘bois noir’ phytoplasma, tuf type-b in 15 symptomatic grapevine samples collected in all surveyed areas.

Genetic diversity of European phytoplasmas of the 16SrV taxonomic group and proposal of ‘Candidatus Phytoplasma rubi’

In addition to the grapevine flavescence dorée phytoplasmas, other members of taxonomic group 16SrV phytoplasmas infect grapevines, alders and species of the genera Clematis and Rubus in Europe. In order to investigate which phytoplasmas constitute discrete, species-level taxa, several strains were analysed by comparing their 16S rRNA gene sequences and a set of five housekeeping genes. Whereas 16S rRNA gene sequence similarity values were >97.5 %, the proposed threshold to distinguish two 'Candidatus Phytoplasma' taxa, phylogenetic analysis of the combined sequences of the tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genetic loci showed that two discrete phylogenetic clusters could be clearly distinguished. The first cluster grouped flavescence dorée (FD) phytoplasmas, alder yellows (AldY) phytoplasmas, Clematis (CL) phytoplasmas and the Palatinate grapevine yellows (PGY) phytoplasmas. The second cluster comprised Rubus stunt (RS) phytoplasmas. In addition to the specificity of the insect vector, the Rubus stunt phytoplasma contained specific sequences in the 16S rRNA gene. Hence, the Rubus stunt phytoplasma 16S rRNA gene was sufficiently differentiated to represent a novel putative taxon: 'Candidatus Phytoplasma rubi'.

Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir

Bois noir is a grapevine yellows disease that is gaining importance in many regions of Europe. The Bois noir phytoplasma (“Candidatus Phytoplasma solani”) was shown to be transmitted by the planthopper Hyalesthes obsoletus, which normally feeds on herbaceous weeds, and occasionally also on grapevines. Three subtypes of the Bois noir phytoplasma have been described and were shown to be associated with distinctive host plants. In this study, we developed a novel and rapid real-time PCR allelic discrimination assay for the distinction of the two major Bois noir phytoplasma subtypes, VK type I and II. Two TaqMan probes carrying different fluorescent dyes were designed to specifically bind to a polymorphism characteristic for the two Bois noir phytoplasma subtypes, thereby allowing discriminative amplification in a single-tube and single-step assay. A total of 259 bois noir-positive grapevine samples collected over 5 years were analysed using the conventional PCR-RFLP method and our newly developed TaqMan allelic discrimination assay. 257 out of 259 samples could be typed with the TaqMan method, compared to 200 out of 259 samples when using the conventional method. The overall concordance of the two methods was 100%. Our newly developed TaqMan assay represents a useful tool for fast and reliable determination of Bois noir phytoplasma subtypes in infected grapevine, insect vector, and host plant samples. The test is suitable for high-throughput analysis and will thereby facilitate further characterisation of Bois noir epidemiology.

Grapevine flavescence dorée phytoplasma

Grapevine flavescence dorée phytoplasma

Genetic variability amongst isolates of Australian grapevine phytoplasmas

Heteroduplex mobility assay (HMA) was used to determine if genetic variability existed amongst isolates of Australian grapevine yellows (AGY) and the Buckland Valley grapevine yellows (BVGY) phytoplasmas. Genetic variability was detected in two isolates of AGY phytoplasma from the same grapevine when they were compared with an AGY phytoplasma standard using HMA of the elongation factor Tu (tuf) gene. HMA of the tuf gene also showed that an isolate of papaya die back (PDB) phytoplasma, which is closely related to AGY phytoplasma, was different from the AGY phytoplasma standard. The two AGY variant phytoplasmas and PDB phytoplasma were indistinguishable from each other when they were compared using HMA of the tuf gene. No variability was observed amongst isolates of BVGY phytoplasma using HMA of the tuf gene.

The Distribution of Grapevine Yellows Disease Associated with the Buckland Valley Grapevine Yellows Phytoplasma

AbstractThree blocks of Chardonnay in one vineyard in the Buckland Valley of Victoria, Australia, were surveyed over 4 years for grapevine yellows disease (GYd). Buckland Valley grapevine yellows phytoplasma (BVGYp) was the only phytoplasma detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in GYd affected grapevines. GYd affected many grapevines and was characterized by remission of disease, some recurrence and occurrences in previously unaffected grapevines. A regional survey of the Buckland Valley indicated that both GYd and BVGYp occurred in the same restricted grape growing area. Within this area, BVGYp was detected in two vineyards that had been established using planting material from different sources. One could therefore speculate that BVGYp was present in these grapevines as a result of aerial transmission and was not present in the original planting material.

Detection and Characterization of an Elm Yellows (16SrV) Group Phytoplasma Infecting Virginia Creeper Plants in Southern Florida.

The polymerase chain reaction (PCR) employing phytoplasma-specific ribosomal RNA primer pair P1/P7 consistently amplified a product of expected size (1.8 kb) from 29 of 36 symptom-less Virginia creeper (Parthenocissus quinquefolia) plants growing in southern Florida. Restriction fragment length polymorphism analysis of P1/P7-primed PCR products indicated that most phytoplasmas detected in Virginia creeper were similar to phytoplasmas composing the elm yellows (16SrV) group. This relationship was verified by reamplification of P1/P7 products using an elm yellows (EY) group-specific rRNA primer pair fB1/rULWS1. rDNA products (1,571 bp) were generated by group-specific PCR from 28 phytoplasma-positive plants and 1 negatively testing plant identified by earlier P1/P7-primed PCR. Analysis of 16S rDNA sequences determined the Virginia creeper (VC) phytoplasma to be phylogenetically closest to the European alder yellows (ALY) agent, an established 16SrV-C subgroup strain. However, presence or absence of restriction sites for endonucleases AluI, BfaI, MspI, RsaI, and TaqI in the 16S rRNA and 16-23S rRNA intergenic spacer region of the VC phytoplasma collectively differentiated this strain from ALY and other 16SrV group phytoplasmas. Failure to detect the VC phytoplasma by PCR employing nonribosomal primer pair FD9f/FD9r suggests that this newly characterized agent varies from known European grapevine yellows (flavescence dorée) phyto-plasmas previously classified as 16SrV subgroup C or D strains.

Effect of phytoplasmal infection on concentration and translocation of carbohydrates and amino acids in periwinkle and tobacco

Source and sink tissue of Catharanthus roseus (periwinkle) infected with the grapevine yellows (GY), apple proliferation (AP), or ash yellows (ASHY) phytoplasma and of Nicotiana tabacum (tobacco) infected with the AP phytoplasma, were examined for concentrations of glucose, fructose, sucrose, starch and total amino acids. Phytoplasmal infection caused growth inhibition and severe yellowing in both plants, the GY phytoplasma being more virulent than the AP and ASHY agents in periwinkle. Leaf size in periwinkle was greatly reduced by the ASHY phytoplasma with little or no effect caused by either the AP or GY agents. Both phytoplasma strain and host plant had an influence on the observed results. In source leaves of periwinkle and tobacco, infection mostly led to a marked increase in soluble carbohydrates and starch. Increase was smallest in periwinkle affected by ASHY. During a dark period of 42 h, carbohydrate levels tended to decrease in healthy plants whereas they remained high in diseased plants. In sink leaves of periwinkle, infections by the GY and AP phytoplasmas led to a marked decrease in sugar whereas starch levels were either only slightly or not affected at all. Infection by the ASHY phytoplasma led to a smaller decrease of sugars and an increase in starch. In tobacco sink leaves, sugars remained largely unchanged whereas starch increased after infection with the AP phytoplasma. In periwinkle roots, soluble carbohydrates were not markedly altered but starch was considerably reduced, in the GY and AP phytoplasmas in particular. In tobacco roots, there was a general decrease in soluble carbohydrates and starch following infection by the AP phytoplasma. The amino acid concentrations were not altered by infection, except that source and sink leaves of ASHY-infected periwinkles and source leaves of AP-infected tobacco showed higher levels of amino acids than healthy plants. The results are discussed with respect to the effect of phytoplasmas on phloem transport, source leaf phenotype and sink growth.