Stolbur Phytoplasma Research Articles

Molecular identification of the Hyalesthes species (Hemiptera: Cixiidae) occurring in vineyard agroecosystems

The insect family Cixiidae includes economically important vectors of plant pathogens. Hyalesthes obsoletus transmits the stolbur phytoplasma (16SrXII‐A genetic group) associated with a serious grapevine yellows disease known as Bois Noir (BN). Other cixiids are currently suspected to be BN vectors, and the correct identification of Hyalesthes species present in vineyards is essential for epidemiological surveys. This study aims to discriminate between H. obsoletus, Hyalesthes luteipes and Hyalesthes scotti by integrating their morphological descriptions with new molecular assays. The sole amplification of a ribosomal internal transcribed spacer 2 (ITS2) region was enough to discriminate between H. luteipes and H. scotti, which are externally indistinguishable. Alternatively, PCR–restriction fragment length polymorphism (RFLP) assays carried out on the mitochondrial cytochrome oxidase I gene (COI) with TaqI provided species‐specific profiles for all three Hyalesthes species. These molecular identification keys proved to be fast and reliable and can contribute to broaden the current handful of entomologists involved in cixiid identification. Furthermore, these markers can also be successfully applied to nymphs, whose morphological distinctive traits are unavailable. The identification of nymphs greatly extends the vector monitoring period and allows the unambiguous association with the actual host plants. Thus, these molecular tools will help monitoring activities and the rational control of potential stolbur vectors. The same ITS2 and COI sequences have been analysed at an intraspecific level for H. obsoletus to identify possible different populations involved in different epidemiological cycles of BN. Both markers confirmed a high degree of genetic conservation within the species and could not characterise populations living on different host plants and transmitting different phytoplasmas strains.

First report of a stolbur phytoplasma associated with witches’ broom of Japanese spindle (Euonymus japonicus)

Japanese spindle trees (Euonymus japonicus) are evergreen shrubs grown for hedges in parks. Recently, diseased trees in Tehran (Iran) were observed with symptoms of little leaves and a bushy appearance suspected to be caused by a phytoplasma. Since the trees showed witches’ broom in one or more of their branches, the name ‘Japanese spindle witches’ broom’ (JSWB) was adopted. In October 2008, samples from seven affected trees were collected in Tehran and the total DNA was extracted by the CTAB procedure (Ahrens & Seemuller, 1992). Amplification of the 16S rDNA was done by nested PCR using generic phytoplasma primers specific to the 16S rRNA gene. The first primer pair was P1/P7 while the nested primers were R16F2 and R16R2 (Deng & Hiruki, 1991; Smart et al., 1996). For positive controls, DNA from alfalfa witches’ broom and safflower phyllody were utilized. DNA from healthy trees served as a negative control. All seven samples gave the expected phytoplasma specific PCR band of 1250 bp, corresponding to those of the positive controls, while healthy trees gave no bands. Amplified DNA products of nested PCR from two samples were isolated and directly sequenced with the forward and reverse primers R16F2 and R16R2. The BLAST analysis of the sequence obtained confirmed that the spindle witches’ broom phytoplasma belongs to the 16SrXII-A (stolbur) group. The sequence was deposited at GenBank (Accession No. GQ273961.) In Iran the stolbur group of phytoplasmas has been detected in potatoes, plums, peaches and almonds (Salehi et al., 2005), and the sequence of JSWB shows 99% homology to the Iranian potato purple top phytoplasmas (EU661607). Since the 16S rDNA sequence of JSWB also shows 99% homology to Bois Noir (BN), a potential grapevine yellows pathogen, grapevines in Iran may be the next target (Karimi et al., 2009). This is the first report of a phytoplasma infecting Japanese spindle trees.

Leafhoppers and cixiids in phytoplasma-infected carrot fields: Species composition and potential phytoplasma vectors

The first molecular analysis of samples collected in southern Backa (Serbia) confirmed the presence of aster yellows (16SrI) and stolbur phytoplasmas (16SrXII) in insects belonging to the family Cicadellidae, as well as in carrot plants where the insects were collected. A correct identification of the phytoplasmas and their vectors is essential to arrange effective control strategies to prevent diseases associated with phytoplasmas from spreading to carrots and other vegetable crops. In order to enhance knowledge about insect vectors of aster yellows and stolbur phytoplasmas in Serbia, Cicadellidae and Cixiidae (Homoptera Auchenorrhyncha), the most common vectors of these phytoplasmas, were monitored in southern Backa during 2008. Adults leaf- and planthoppers were collected and identified at species level using standard entomological methods, and tested for phytoplasma presence by means of PCR/RFLP. A total of 13 insect species of Cicadellidae were identified, as follows: a) three species of the subfamily Agallinae: Anaceratagallia ribauti (Ossiannilsson), Anaceratagallia venosa (Fourcroy), and Anaceratagallia laevis (Ribaut); b) seven species of the subfamily Deltocephalinae: Psammotettix confinis (Dahlbom), Psammotettix striatus (Linnaues) Psammottettix alienus (Dahlbom), Macrosteles sexnotatus (Fall?n), Ophiola decumana (Kontkanen), Errastunus ocellaris Fall?n, and Scaphoideus titanus Ball; c) three species of the subfamily Typhlocibinae: Eupteryx atropunctata (Goeze), Eupteryx mellissae Curtis, Zyginidia pullula (Boheman). Female specimens of the genus Euscelis (Deltocephalinae) were also collected, as well as one species of Reptalus quinquecostatus (Dufour) of the family Cixiidae. Stolbur phytoplasmas were detected in A. laevis, A. ribauti, A. venosa, P. striatus, P. confinis and P. alienus. The species: A. laevis, O. decumana, and P. confinis were AY-infected (subgroup 16SrI-A), while subgroup 16SrI-C was found only in one specimen of P. confinis. Since some aster yellows- and stolbur-infected species of the genera Psammotettix and Anaceratagallia (especially P. confinis and A. laevis) were regularly and commonly found in the infected carrot fields during the whole vegetative period, they could play a significant role in transmitting and spreading these pathogens in natural environment.

Detection of XIIA phytoplasma group on cultivar Zupljanka in Zupa vineyard region by RFLP analysis of 16s rDNA sequences

'Bois noir' (BN) is an important grapevine disease associated with phytoplasmas belonging to ribosomal subgroup 16SrXII-A. Phytoplasmas cause diseases in several hundred plant species. The number of infected cultivars is growing each year and it is important to follow the spreading of the phytoplasma in the different regions and identify which strains are present in specific regions on specific cultivars. Phytoplasmas are identified and classified based on direct sequencing of phytoplasma 16S rDNA or the 16S to 23S intergenic spacer region, but this approach is not always practical when a large number of unknown phytoplasmas is to be analyzed. Classification by RFLP analysis has provided a simple and rapid method that can be used to differentiate and identify a large number of unclarified phytoplasmas. Our objective was to investigate presence of phytoplasmas of 16SrXII-A group (Stolbur) in Zupa vineyard region. Detection was based on RFLP analysis of 16s rDNA sequences using four restriction enzymes: Tru1I, AluI, KpnI and TaqI. We identified phytoplasmas of XIIA group on two of three investigated cultivars (Zupljanka and Frankovka, but not on Plovdina) in the Zupa vineyard regions (Gornje Rataje and Tules locality). This is the first report of Stolbur phytoplasma on cv. Zupljanka in Zupa region.

Occurrence and Distribution of Grapevine Yellows Caused by Stolbur Phytoplasma in Montenegro

AbstractIn August 2008, grapevine yellows (GY)‐like symptoms were observed in vineyards near Podgorica, Montenegro. A survey of the grape growing area of Montenegro was conducted to identify and determine the distribution of phytoplasmas associated with grapevine. Phytoplasmas were detected in symptomatic vine plants using PCR with universal primers for amplification of the phytoplasma 16S rRNA gene. From the 42 plants sampled, 36 were positive for the presence of phytoplasmas. RFLP profiles of the 16S rDNA of all phytoplasma positive isolates tested indicated that they belong to 16SrXII‐A phylogenetic group. PCR with stolbur specific primers confirmed the presence of stolbur phytoplasma in grapevine samples from Montenegro. Sequence and RFLP analysis of the tuf gene indicated the presence of the tufAY‐a and tufAY‐b types of stolbur phytoplasma. To our knowledge, this is the first report of phytoplasma infection of grapevine in Montenegro.

The sensory structures of the antennal flagellum in Hyalesthes obsoletus (Hemiptera: Fulgoromorpha: Cixiidae): A functional reduction?

Despite their relevance as harmful pests on plants of economic importance, Hemiptera Fulgoromorpha have been poorly studied as regards their antennal sensory structures. In particular, the flagellum has been neglected and, therefore, to date there are no data on its structural organization and sensory equipment. In order to fill this gap, we carried out a study on the sensillum types and distribution on the flagellum of the planthopper Hyalesthes obsoletus Signoret, an efficient vector of the stolbur phytoplasma, the cause of various crop diseases. In this cixiid species the antenna is composed of three segments, the scape, an enlarged pedicel and a long flagellum. This latter is made of a single segment and presents a basal, bulb-like enlargement from which two processes arise, a short spur and a long arista. Combining scanning electron microscopy, transmission electron microscopy and focused ion beam investigations, we discovered the presence of a total number of 6 sensilla, belonging to 4 different types: a single scolopidium extending from the bulb to the arista, three sensilla styloconica within the cuticular spur and two different sensilla coeloconica inside the bulb. As far as structural data can suggest, these sensilla might be involved in the perception of mechanical stimuli (possibly air-borne vibrations), temperature and humidity variations and CO 2 concentration. The strong reduction in sensillum number in this species is discussed as possible functional specialization of the flagellum itself. The ultrastructure of the sensilla in the flagellum of a species of Fulgoromorpha is here presented for the first time.

Stolbur Phytoplasma Transmission to Maize by Reptalus panzeri and the Disease Cycle of Maize Redness in Serbia

Maize redness (MR), induced by stolbur phytoplasma ('Candidatus Phytoplasma solani', subgroup 16SrXII-A), is characterized by midrib, leaf, and stalk reddening and abnormal ear development. MR has been reported from Serbia, Romania, and Bulgaria for 50 years, and recent epiphytotics reduced yields by 40 to 90% in South Banat District, Serbia. Potential vectors including leafhoppers and planthoppers in the order Hemiptera, suborder Auchenorrhyncha, were surveyed in MR-affected and low-MR-incidence fields, and 33 different species were identified. Only Reptalus panzeri populations displayed characteristics of a major MR vector. More R. panzeri individuals were present in MR-affected versus low-MR fields, higher populations were observed in maize plots than in field border areas, and peak population levels preceded the appearance of MR in late July. Stolbur phytoplasma was detected in 17% of R. panzeri adults using nested polymerase chain reaction but not in any other insects tested. Higher populations of R. panzeri nymphs were found on maize, Johnsongrass (Sorghum halepense), and wheat (Triticum aestivum) roots. Stolbur phytoplasma was detected in roots of these three plant species, as well as in R. panzeri L(3) and L(5) nymphs. When stolbur phytoplasma-infected R. panzeri L(3) nymphs were introduced into insect-free mesh cages containing healthy maize and wheat plants, 89 and 7%, respectively, became infected. These results suggest that the MR disease cycle in South Banat involves mid-July transmission of stolbur phytoplasma to maize by infected adult R. panzeri. The adult R. panzeri lay eggs on infected maize roots, and nymphs living on these roots acquire the phytoplasma from infected maize. The nymphs overwinter on the roots of wheat planted into maize fields in the autumn, allowing emergence of phytoplasma-infected vectors the following July.

Molecular Identification of Stolbur Phytoplasma Associated with Red Clover Dwarf Disease Symptoms

Abstract Trifolium pratense plants exhibiting dwarf symptoms without flower abnormalities were frequently observed in the breeding nurseries in North Moravia, Czech Republic. Direct and nested polymerase chain reaction with phytoplasma‐specific primers and subsequent restriction fragment length polymorphism analyses of 16S rDNA, as well as sequence analyses of the 16S–23S ribosomal operon, identified phytoplasmas belonging to the ribosomal subgroup 16SrXII‐A. Identical analyses of the dnaB gene and the transcription factor tuf gene were used for further differentiation. The primer/enzyme‐combination of TufAYf,r/HpaII revealed the grouping of the phytoplasmas into stolbur tuf‐type II in all examined samples. Stolbur phytoplasmas have not been reported previously in red clover.

GENETIC VARIABILITY OF STOLBUR PHYTOPLASMA IN ANNUAL CROP AND WILD PLANT SPECIES IN SOUTH MORAVIA

SUMMARY The genetic diversity of stolbur phytoplasma isolates was assessed by different molecular typing methods in a wide range of plant hosts in a limited geographical area of the Czech Republic. Plants of Apium graveolens, Capsicum annuum, Solanum lycopersicum, Solanum tuberosum, Amaranthus retroflexus, Calystegia sepium, Cirsium arvense, Convolvulus arvensis, and Datura stramonium displaying symptoms of proliferation, discoloration, and malformation of flower were collected in vegetable crop plots in the vicinity of the village of Lednice (south Moravia) and examined for the presence of phytoplasmas. Using 16S rDNA amplification and RFLP assays, stolbur phytoplasma was detected in some of the symptomatic plants. The genotype of the phytoplasma isolates detected was investigated by restriction pattern or sequence analysis of two non-ribosomal house-keeping genes (tuf and secY) and a recently characterized polymorphic gene (vmp1). According to tuf genotyping, all isolates were of the tufAY-b genotype sensu Langer and Maixner (2004), a genotype known to be associated with bindweed (C. arvensis) reservoirs. A finer isolate differentiation was obtained with vmp1 genotyping as four genetic variants carrying different vmp1 RFLP patterns were found, especially in C. arvensis. In addition, preliminary sampling in other south Moravian localities and secY sequence analyses diclosed also the presence of stolbur isolates different from those previously reported in binweed (C. arvensis) and grapevine.

The incidence of stolbur disease and associated yield losses in vegetable crops in South Moravia (Czech Republic)

The study was performed at a vegetable farm from 2006 to 2008 in the intensive horticultural area of Lednice (South Moravia, Czech Republic), where a stolbur (phytoplasma) epidemic had occurred. The study showed that the incidence of stolbur disease reached 15% in both tomato ( Lycopersicon esculentum) and pepper ( Capsicum annuum), and up to 6.7% in celeriac ( Apium graveolens). There were significant yield losses in the stolbur-affected plants; total yield losses were up to 60% in tomato, 93% in pepper, and 100% in celeriac. The mean yield was significantly decreased in stolbur-affected plants, compared to healthy plants ( i.e. from 42.79 to 17.21 fruits per plant in tomatoes; from 10.11 to 0.74 fruits per plant in peppers). In the locality studied, it was mainly the weed plants Convolvulus arvensis and Cirsium arvense (which were frequently interspersed among the crops), which tested positive for the stolbur phytoplasma and might have provided a reservoir for the phytoplasma infection.

Characterization of Bois Noir Isolates by Restriction Fragment Length Polymorphism of a Stolbur-Specific Putative Membrane Protein Gene

Bois noir phytoplasma (BNp), widespread in wine-producing areas of Europe and endemic in France and Italy, is classified in the 16SrXII-A subgroup, whose members are referred to as Stolbur phytoplasmas. The 16S rDNA gene of Stolbur phytoplasma shows low variability, and few non-ribosomal genes are available as markers to assess variation among isolates. We used the Stolbur-specific stol-1H10 gene, encoding a putative membrane-exposed protein, to investigate genetic diversity of French and Italian BNp isolates from plants and insects. Amplification of stol-1H10 from infected grapevines, weeds, and Hyalesthes obsoletus produced fragments of three sizes, and restriction fragment length polymorphism analysis divided these amplicons further into 12 profiles (V1 to V12). French BNp isolates were more variable than Italian ones, and different profiles were present in infected grapevines from France and Italy. Isolate V3, most abundant among Italian affected grapes but present among French ones, was found in one Urtica dioica sample and in all H. obsoletus collected on this species. Four Italian-specific profiles were represented among infected Convolvulus arvensis, the most frequent of which (V12) was also detected in H. obsoletus collected on this species. Most of the variability in the stol-1H10 sequence was associated with type II on the tuf gene.

Occurrence of Stolbur phytoplasma in the vector Hyalesthes obsoletus, herbaceous host plants and grapevine in South Tyrol (Northern Italy).

Bois noir (BN) is a grapevine yellows disease caused by a phytoplasma of the Stolbur group (16SrXII-A). The planthopper Hyalesthes obsoletus is known to be the principal vector and can accidentally transmit the phytoplasma from its herbaceous host plants to grapevine ( Vitis vinifera ). Due to the increasing incidence of BN over the last decade, a monitoring study was conducted in South Tyrol (Northern Italy). Over a period of up to four years, 659 insect vector samples, 516 herbaceous plants of 41 potential host plant species as well as 56 grapevine samples from BN-affected vineyards were tested for the presence of the Stolbur phytoplasma using a nested PCR procedure. In addition, a recently developed TaqMan allelic discrimination assay was employed to determine different subtypes of BN in infected samples. The Stolbur phytoplasma could be detected in all three sample types analysed, and was shown to belong to two different subtypes, VK type I and VK type II. In most vineyards one subtype was found to be predominant. The average infection rate of H. obsoletus amounted to 24.1 %. Analysis of herbaceous plants revealed that 25.1 % of the Convolvulus arvensis samples tested positive for the BN phytoplasma, as well as 4.5 % of the Urtica dioica samples. Taken together, our results underline the role of these two species commonly found in the undergrowth vegetation of South Tyrolean vineyards as an important reservoir of the Stolbur phytoplasma.

Striking diversity of vmp1, a variable gene encoding a putative membrane protein of the stolbur phytoplasma.

Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the "Candidatus Phytoplasma asteris" genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The vmp1 gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to vmp1 in the genomes of nine StolP isolates. vmp1 is variable in size, and eight different vmp1 RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of vmp1 sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates. vmp1 appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions.

New 16Sr subgroups and distinct single nucleotide polymorphism lineages among grapevine Bois noir phytoplasma populations

AbstractBois noir (BN) is an insect‐transmitted grapevine yellows disease caused by phytoplasmas belonging to the stolbur subgroup 16SrXII‐A. In Italy, increasing prevalence of stolbur phytoplasma strains in vineyards suggests progressive spread of the disease and potential for heavy impacts on the wine industry. In this study, we investigated the genetic diversity of stolbur phytoplasma strains in BN phytoplasma populations. Nucleotide sequences of16S rRNAgenes from stolbur phytoplasma strains affecting vineyards in the Lombardy region of Italy and stolbur phytoplasma 16S rDNA sequences retrieved from GenBank were subjected to virtual restriction fragment length polymorphism analysis. Calculation of virtual restriction similarity coefficients revealed the presence of new subgroups in group 16SrXII (stolbur phytoplasma group). Representative strains of confirmed new subgroups 16SrXII‐F (XII‐F) and XII‐G and tentative new subgroups XII‐A1 through XII‐A19, XII‐H, XII‐I, and XII‐J as well as known subgroup XII‐A were from grapevines; strains representing three additional tentative new subgroups (XII‐K, XII‐L and XII‐M) were from other plant hosts. Nucleotide sequence alignments identified no less than nine genetically distinct 16S rDNA single nucleotide polymorphism lineages from grapevine, indicating a high degree of genetic heterogeneity within BN phytoplasma populations. The findings open new opportunities for in‐depth studies of the distribution of grapevine‐associated 16SrXII phytoplasma strains in weeds, insect vector populations and grapevines from vineyards located in different geographic areas.

Ability of Reptalus quinquecostatus (Hemiptera: Cixiidae) to inoculate stolbur phytoplasma to artificial feeding medium

AbstractLaboratory trials were carried out on wild individuals of Reptalus quinquecostatus (Cixiidae), a potential vector of stolbur phytoplasma to grapevine, to assess its ability to inoculate the phytoplasma in artificial feeding medium. Seventy‐seven specimens of the cixiid were tested on a sucrose–TE (Tris–ethylenediaminetetraacetic acid) diet and 62 of them survived less than 24 h. Polymerase chain reaction (PCR) assays performed on the insect bodies detected the presence of stolbur phytoplasma, with an infection rate of 32.5%. Restriction fragment length polymorphism analysis of the tuf gene, amplified by PCR, revealed Vergilbungskrankheit type I (VK‐I) in 20 specimens, VK‐II in 4 specimens and both types in 1 specimen. Ten of the 25 infected R. quinquecostatus specimens successfully inoculated VK‐I in the sucrose solution, that is, a 40% inoculation efficiency despite the brief survival. The results indicate that R. quinquecostatus is a competent species to transmit the stolbur phytoplasma in artificial conditions. The repeated observation of adults feeding on grapevine strengthens the hypothesis that the species is a vector of stolbur phytoplasma to this plant.

Transmission of a Stolbur Phytoplasma by the Agalliinae Leafhopper Anaceratagallia ribauti (Hemiptera, Auchenorrhyncha, Cicadellidae)

AbstractSeventeen Auchenorrhyncha species were studied for their ability to transmit stolbur phytoplasma. Adult insects were collected alive in Bois Noir infected vineyards, transferred to Vicia faba seedlings (two leaf stage) in cylindrical pot cages and kept in a growth chamber at 23°C (L16:D8). After 5 weeks, plants were inspected visually and sampled for PCR analysis. Anaceratagallia ribauti, Dryodurgades reticulatus and Euscelis incisus were also reared in the laboratory. Insects trapped in the vineyard were allowed to oviposit on V. faba seedlings. Hatching nymphs were transferred to cubical breeding cages containing potted V. faba seedlings and stolbur‐infected Convolvulus arvensis collected in the vineyard. Plants in these cubical cages were routinely tested by PCR for stolbur infections. Adults raised in these cubical cages were subjected to further pot cage experiments with V. faba seedlings. In five of 40 experiments with field‐trapped A. ribauti, stolbur transmission was observed. Laboratory‐reared A. ribauti transmitted stolbur phytoplasma to seven of 23 broad beans in the cubical breeding cages. Laboratory‐reared A. ribauti adults transmitted stolbur phytoplasma in one of 13 pot cage experiments. No transmission of stolbur phytoplasma with any other Auchenorrhyncha species was observed. Furthermore, we characterized Austrian stolbur isolates by Restriction Fragment Length Polymorphism analysis of the tuf gene. Plant samples were collected in 17 symptomatic vineyards all over eastern Austria. Vicia faba plants from transmission experiments were also analysed. All isolates belonged to tuf‐type II.

Study of the transmission of stolbur phytoplasma to different crop species, by Macrosteles quadripunctulatus

AbstractIn an epidemiological study conducted on commercial agricultural plots affected by stolbur phytoplasma in Northern and Central Spain, different species of leafhoppers and planthoppers were identified as potential vectors of the phytoplasma. They included individuals of Macrosteles quadripunctulatus infected by stolbur phtytoplasma in most of the locations. The potential of this species as a vector of stolbur was evaluated in this work. The transmission trials were carried out on healthy plants of Catharanthus roseus (periwinkle), Lycopersicon esculentum (tomato), Daucus carota (carrot), Lactuca sativa (lettuce) and Vitis vinifera (grapevine). The first symptoms of infection in these plants were observed 2 weeks after the inoculation period in tomato and periwinkle, and after 4 weeks in carrot. Only one of five grapevines showed phytoplasma symptoms. PCR analysis was used to verify the ability of M. quadripunctulatus in transmitting stolbur phytoplasma in the plant species tested. The phytoplasma was not detected in lettuce or in the healthy control plants. Studies of stolbur transmission to insect‐feeding medium were also conducted and indicated that M. quadripunctulatus acquires and was capable of transmitting the phytoplasma after it fed during a single day on infected plants followed by a 19‐day latent period on healthy plants.

Insect Vectors (Hemiptera: Cixiidae) and Pathogens Associated with the Disease Syndrome "Basses Richesses" of Sugar Beet in France.

The syndrome "basses richesses" (SBR) is a disease of sugar beet in eastern France associated with two phloem-restricted, nonculturable plant pathogens: a stolbur phytoplasma and a γ-3 proteobacterium, here called SBR bacterium. Three planthopper (Hemiptera: Cixiidae) species were found to live near and within sugar beet fields in eastern France: Cixius wagneri, Hyalesthes obsoletus, and Pentastiridius leporinus. The role of these planthoppers in spreading the two pathogens to sugar beet was studied. Based on its abundance and high frequency of infection with the SBR bacterium, P. leporinus was considered to be the economic vector of SBR disease. C. wagneri, the primary vector of 'Candidatus Phlomobacter fragariae' to strawberry in western France, also was found to be infected by the SBR bacterium and to transmit the pathogen to sugar beet. Neither C. wagneri nor P. leporinus were infected by stolbur phytoplasma. Populations of H. obsoletus living on bindweed (Convolvulus arvensis) and nettle (Urtica dioica) collected near sugar beet fields did not carry the SBR bacterium, but were highly infected with two restriction fragment length polymorphism-differentiable stolbur phytoplasma isolates. In transmission assays, only the bindweed phytoplasma isolate was transmissible to and pathogenic on sugar beet. When compared with controls, the bindweed stolbur phytoplasma and SBR bacterium similarly reduced the biomass of sugar beet plants, but the phytoplasma caused greater reductions in taproot biomass and sugar content than the SBR bacterium.

First Detection of Stolbur Phytoplasma in Grapevines (Vitis vinifera cv. Merlot) Affected with Grapevine Yellows in Bulgaria

AbstractBetween 2003 and 2005, a survey was conducted throughout the grape‐growing regions of Bulgaria to identify possible infection with grapevine yellows diseases, especially Flavescence dorée (FD). The samples were checked for phytoplasmas and viruses inducing similar symptoms in the Central Laboratory for Plant Quarantine. To confirm stolbur phytoplasma infection of grapevine, a multiplex nested‐PCR assay for direct detection of FD and stolbur phytoplasmas was used. Infection of grapevine with phytoplasma was detected. The disease is very common disease in Bulgaria on tomatoes, potatoes and other crops. Monitoring is being continued. This is the first report of phytoplasma‐infected grapevine in Bulgaria.

First Report of Bois Noir Phytoplasma in Grapevine in Canada.

During the summer and fall of 2006, a survey was done to detect European phytoplasmas of quarantine significance in Canadian vineyards. This survey was developed as one of the 2006 import requirements for grapevine nursery stock from Europe. This addresses the increased concerns regarding inadvertent phytoplasma introductions. Grapevines imported in 2006 and established grapevines were observed for symptoms typical of those associated with diseases caused by phytoplasmas on grapevine. Samples were tested from 155 grapevines. One plant, located in the lower Okanagan Valley, British Columbia, tested positive by a modified real-time PCR assay and TaqMan probe targeting the 16S region of the ribosomal RNA gene (1), which detects a wide variety of known phytoplasmas. The sample was further analyzed and found to be positive by conventional PCR with the phytoplasma-specific primers, P1/P7 (3), and Stolbur specific primers, STOL11f2/r1 (2). Additional PCR tests with primers specific to flavescence doree (FD9f/r) (2) and western X disease (P1/W INT) (3) were negative. These phytoplasmas are also known to infect grapevine. The approximate 1,800-bp fragment obtained with P1/P7 was sequenced (GenBank Accession No. EU086529) and found to have 99.7% nucleotide sequence identity to the Stolbur STOL #11 isolate (GenBank Accession No. AF248959) originally isolated from eastern Europe. This was the highest match to any available phytoplasma sequence obtained and indicates that the phytoplasma in the British Columbian sample is an isolate of bois noir, a pest of quarantine significance to Canada. Additional phylogenetic analysis using CLUSTAL W (Lasergene; DNASTAR, Madison, WI) confirmed this result. The presence and identity of the phytoplasma was confirmed from a second tissue sample that was analyzed by PCR and sequenced using the same test procedures as for the first sample, with identical results. The bois noir phytoplasma belongs to the stolbur group (16SrVII) with the principal vector being a cixiid planthopper. Stolbur phytoplasmas cause diseases in other crops, but bois noir disease is caused by a specific member of that group and is the only stolbur phytoplasma known to infect grapevines in Europe. The infected grapevine was from a lot of 1,965 plants of Grenache clone 70 on rootstock 3309 clone 143 that was imported from Europe in 2006. All plants in this importation have been destroyed. This phytoplasma has not been detected in any other grapevines in Canada. Additional import conditions requiring hot water treatment of European vines have been implemented for 2007. Further survey work for phytoplasma in grapevine will continue.